Manufacture, formulation and dosing of apraglutide

ABSTRACT

The present disclosure relates to methods of making, formulating and administering GLP-2 analogs.

RELATED APPLICATIONS

This application is a Continuation of International Patent ApplicationNo. PCT/US2021/036655, filed Jun. 9, 2021, which claims priority to, andthe benefit of, U.S. Provisional Application No. 63/036,507, filed Jun.9, 2020, U.S. Provisional Application No. 63/074,119, filed Sep. 3,2020, and U.S. Provisional Application No. 63/157,083, filed Mar. 5,2021. The contents of each of the aforementioned patent applications areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 21, 2021, isnamed “VECT-002_C01US_SeqList.txt” and is about 2 KB in size.

TECHNICAL FIELD

The present disclosure relates to manufacture, formulation and dosing ofapraglutide.

BACKGROUND

Glucagon-like peptide 2 (GLP-2) is a 33-amino-acid peptide released fromthe post-translational processing of proglucagon in the enteroendocrineL cells of the intestine.

We contemplate using apraglutide to treat intestinal disorders ordysfunction. In one embodiment we contemplate treatment of short bowelsyndrome. Short bowel syndrome is a malabsorptive conditioncharacterized by extreme reduction in functional intestinal length mostcommonly as a result of surgical resection due to mesenteric ischemia orInflammatory Bowel Disease (IBD) although other etiologies are alsopresent. SBS with intestinal failure (SBS-IF) is defined as thereduction of gut function below the minimum necessary for the absorptionof macronutrients and/or fluids and electrolytes, such that intravenoussupplementation is required to maintain health and/or growth. Inpatients with SBS-IF parenteral support (PS) delivered through a centralvenous catheter is needed to maintain an adequate fluid, energy,electrolytes, trace elements, vitamins, and nutrient balance. There isspectrum of SBS-IF patients from those with stoma and nocolon-in-continuity (CIC) requiring large PS volumes to those with CICwhich require lower PS volumes. As a consequence of this spectrum,decreased dependency on PS can be demonstrated in a variety of outcomesdepending on anatomy, including PS volume reduction, days off PS, orachieving enteral autonomy. After surgical resection, the remainingintestine goes through a process called intestinal adaptation by whichit increases its absorptive capacity to compensate for its reducedlength. It has been demonstrated that this process of intestinaladaptation can be enhanced by administering glucagon-like peptide 2(GLP-2) or more stable analogues with extended half-lives such asteduglutide and apraglutide. Because of the physiological role of thecolon, patients with CIC can manage fluid balance much better thanpatients with no functional colon and have lower parenteral supportvolumes at baseline compared with stoma patients. PS is defined as anyintravenous infusion that contains fluids and electrolytes and may ormay not include parenteral nutrition (PN). Parenteral nutrition isdefined as PS that includes protein, carbohydrate, fat, vitamins and/ortrace elements. Patients with chronic intestinal failure due to benigndisease have a high probability of long-term survival on PS.

GLP-2 has attracted considerable attention as a therapeutic agent forintestinal injury since its identification as a potent stimulator ofmucosal epithelial proliferation. Preliminary trials in patients withshort bowel syndrome have produced improvements in intestinal absorptionof both fluids and nutrients. GLP-2 induces significant growth of thesmall intestinal mucosal epithelium. It also slows intestinal transit.Naturally occurring GLP-2 is, however, not a suitable drug candidate, asit is rapidly degraded by peptidases (e.g. DPP IV). As a result, GLP-2has a very short half-life (t½=10 min. in humans) and rapid clearance(CL).

A method is needed to generate a high yielding and substantially pureGLP-2 analog peptide with improved pharmacokinetic properties. Further,there is a need for a GLP-2 analog peptide composition that issubstantially pure e.g. substantially free from impurities. The presentdisclosure is directed to satisfying this need.

SUMMARY OF THE DISCLOSURE

In one aspect, a novel synthesis of a GLP-2 analog of high purity isdisclosed. In another aspect, a novel sodium salt of a GLP-2 analog isdisclosed. The method may comprise performing solid phase peptidesynthesis (SPPS) on a resin, cleaving the synthesized GLP-2 analogpeptide off the resin and deprotecting the side chains of the peptide bytreating the resin with a solution comprising trifluoroacetic acid(TFA), water, and anisole, performing two purifications usingreversed-phase high performance liquid chromatography (RP-HPLC), andfreeze-drying and packaging the purified peptide powder comprising thesubstantially pure GLP-2 analog peptide. In some embodiments, theapraglutide has a purity of no less than 95%. In some embodiments, theapraglutide has a purity of no less than 97%.

Another aspect of the present disclosure is directed to the GLP-2 analogpeptide having a purity of at least 97%. In some embodiments, the GLP-2analog peptide, such as apraglutide, comprises less than 1% of aDes-Gly⁴ apraglutide impurity; and/or less than 3% of the sum ofAspartimide³ apraglutide, Asp³³-OH apraglutide and Des-Ser⁷ apraglutideimpurities; and/or less than 1% of a [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)]apraglutide impurity.

Another aspect of the present disclosure is directed to the GLP-2 analogpeptide having a purity of at least 97%. In some embodiments, the GLP-2analog peptide, such as apraglutide, comprises less than or equal to 1%of a Des-Gly⁴ apraglutide impurity; and/or less than or equal to 3% ofthe sum of Aspartimide³ apraglutide, Asp³³-OH apraglutide and Des-Ser⁷apraglutide impurities; and/or less than or equal to 1% of a [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] apraglutide impurity.

Another aspect of the present disclosure is directed to the GLP-2 analogpeptide having a purity of at least 97%. In some embodiments, the GLP-2analog peptide, such as apraglutide, comprises less than 1% of the sumof Des-Gly⁴ apraglutide and Aspartimide³ apraglutide impurities, lessthan 1% of D-Aspartimide³ apraglutide impurity, less than 1% of Asp³³-OHapraglutide impurity, less than 1% of Des-Ser⁷ apraglutide impurities,and/or less than 1% of a [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] apraglutideimpurity.

Another aspect of the present disclosure is directed to the GLP-2 analogpeptide having a purity of at least 97%. In some embodiments, the GLP-2analog peptide, such as apraglutide, comprises less than or equal to 1%of the sum of Des-Gly⁴ apraglutide and Aspartimide³ apraglutideimpurities, less than or equal to 1% of D-Aspartimide³ apraglutideimpurity, less than or equal to 1% of Asp³³-OH apraglutide impurity,less than or equal to 1% of Des-Ser⁷ apraglutide impurities, and/or lessthan or equal to 1% of a [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] apraglutideimpurity.

The inventors have recognized that:

i) replacement of Fmoc-protected amino acids: Fmoc-Gln(Trt)-OH,Fmoc-Thr(tBu)-OH and Fmoc-Gly-OH with Fmoc-Gln(Trt)-Thr(ψ^(Me,Me)pro)-OHand Fmoc-Tmb-Gly-OH during Fmoc deprotection and coupling cyclesimproves coupling efficiency and reduces aspartimide formation;ii) the use of oxyma instead of HOBt as the coupling additive duringFmoc deprotection and coupling cycles minimizes peptide oxidation and[Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] apraglutide impurity formation; andiii) during sodium salt conversion by preparative RP-HPLC, the use ofacetonitrile (ACN) for the mobile phase composition, the introduction ofa pH adjustment step, and the removal of a titration step, improvesapraglutide sodium salt conversion and purified apraglutide quality. Inanother aspect, a novel formulation and salt of apraglutide isdisclosed.

According to one embodiment, the GLP-2 analog peptide of the presentdisclosure has the chemical structure:

In another aspect, a novel method of dosing an effective amount ofapraglutide to patients in need thereof is disclosed. In someembodiments, the effective amount comprises between 1 and 10 mg of theapraglutide or a pharmaceutically acceptable salt thereof. In apreferred embodiment, apraglutide or a pharmaceutically acceptable saltthereof is dosed at 2.5 mg once weekly in patients that weigh less than50 kg. In another preferred embodiment, apraglutide or apharmaceutically acceptable salt thereof is dosed at 5.0 mg once weeklyin patients that weigh 50 kg or more. In some embodiments, the methodcomprises administering apraglutide or pharmaceutically acceptable saltthereof intravenously. In other embodiments, the method comprisesadministering apraglutide or pharmaceutically acceptable salt thereofsubcutaneously.

In some embodiments, the method comprises administering the compound orpharmaceutically acceptable salt thereof at a frequency between twicedaily and twice monthly, preferably once weekly.

There is a need for a GLP-2 analog with a longer half-life, such asapraglutide, that can be dosed less than once daily, preferably onceweekly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the peptide sequence and structure for an exemplary GLP-2agonist (apraglutide) that can be used in the methods of the presentdisclosure. Sequences shown in FIG. 1 correspond to SEQ ID NOs: 1-4.

FIG. 2A is a flow diagram schematic of steps 1-2 in a first aspect ofthe present disclosure directed to an improved method of making a GLP-2analog peptide, such as apraglutide (hereafter referred to as ProcessB).

FIG. 2B is a flow diagram schematic of steps 3-6 in a first aspect ofthe present disclosure directed to an improved method of making a GLP-2analog peptide, such as apraglutide (hereafter referred to as ProcessB).

FIG. 3 is flow diagram schematic of a manufacturing process of thepresent disclosure directed to the production of a pharmaceuticalcomposition comprising apraglutide.

FIG. 4 is a diagram schematic of the trial design of the presentdisclosure.

FIG. 5 is a graph showing individual and mean changes from baseline toend of treatment in urine volume output (mL/day). Dashed line denotesthe mean, A denotes the mean change from baseline (standard deviation).The difference in grayscale shows individual patients.

FIG. 6 is a graph showing the changes from baseline in urine volumeoutput (mL/day) where the adjusted mean (points) and 95% CI (blacklines) are from analysis Part A+B.

FIG. 7 is a graph showing individual and mean changes from baseline toend of treatment in urine sodium excretion (mmol/day). Dashed linedenotes the mean, B denotes the baseline, T denotes treatment, A denotesthe mean change from baseline (standard deviation). The difference ingrayscale shows individual patients.

FIG. 8 is a graph showing individual and mean changes from baseline toimmediately after first injection in urine volume output. Dashed linedenotes the mean, B denotes the baseline, T denotes treatment, A denotesthe mean change from baseline (standard deviation). The difference ingrayscale shows individual patients.

FIG. 9 is a graph showing individual and mean changes from baseline toimmediately after first treatment in urine sodium excretion. Dashed linedenotes the mean, B denotes the baseline, T denotes treatment, A denotesthe mean change from baseline (standard deviation). The difference ingrayscale shows individual patients.

FIG. 10A is a graph showing individual changes and mean change in fluidcomposite effect in patients receiving placebo treatment. Fluidcomposite effect, defined as the sum of increase urine production (day27-29), reduction in PS volume and reduction in spontaneous oral fluidintake (day 20-22).

FIG. 10B is a graph showing individual changes and mean change in fluidcomposite effect in patients receiving 5 mg apraglutide. Fluid compositeeffect, defined as the sum of increase urine production (day 27-29),reduction in PS volume and reduction in spontaneous oral fluid intake(day 20-22).

FIG. 10C is a graph showing individual changes and mean change in fluidcomposite effect in patients receiving 10 mg apraglutide. Fluidcomposite effect, defined as the sum of increase urine production (day27-29), reduction in PS volume and reduction in spontaneous oral fluidintake (day 20-22).

FIG. 11 is a graph showing individual and mean changes from baseline toend of treatment in absolute concentrations of plasma L-citrulline.Dashed line denotes the mean, B denotes the baseline, T denotestreatment, A denotes the mean change from baseline (standard deviation).The difference in grayscale shows individual patients.

FIG. 12 is a diagram schematic of the Consolidated Standards ofReporting Trials (CONSORT).

FIG. 13 is a diagram schematic of the Consolidated Standards ofReporting Trials (CONSORT).

FIG. 14 is a graph showing individual and mean changes from baseline toend of treatment in wet weight dietary intake, fecal output, intestinalabsorption and urine production. Bold line denotes the mean, B denotesthe baseline, T denotes treatment, A denotes the mean change frombaseline (standard deviation). Dashed lines show patients with SBS-II.Difference in grayscale shows individual patients.

FIG. 15 is a graph showing individual and mean changes from baseline toend of treatment absorption of potassium, sodium, magnesium and calcium.Bold line denotes the mean, B denotes the baseline, T denotes treatment,A denotes the mean change from baseline (standard deviation). Dashedlines show patients with SBS-II. Difference in grayscale showsindividual patients.

FIG. 16 is a graph showing individual and mean changes from baseline toend of treatment in the energy dietary intake, fecal output andabsorption. Bold line denotes the mean, B denotes the baseline, Tdenotes treatment, A denotes the mean change from baseline (standarddeviation). Dashed lines show patients with SBS-II. Difference ingrayscale shows individual patients.

FIG. 17 is a graph showing individual and mean changes from baseline toend of treatment in absorption of macronutrients. Bold line denotes themean, B denotes the baseline, T denotes treatment, A denotes the meanchange from baseline (standard deviation). Dashed lines show patientswith SBS-II. Difference in grayscale shows individual patients.

FIG. 18 is a graph showing plasma citrulline concentrations (ug/mL) as afunction of time (days) after the first dose of apraglutide. SD=standarddeviation; SC=subcutaneous. Data are arithmetic means (±SD). Weekly SCadministrations of 1, 5 or 10 mg apraglutide were scheduled on days 1,8, 15, 22, 29 and 36.

FIG. 19 is a graph showing the corresponding least square (LS) mean plotfor change from baseline for plasma citrulline concentrations (ug/mL) asa function of time (days) after the first dose of apraglutide.CI=confidence interval; SC=subcutaneous. Data are estimated least squaremeans (95% CI). Weekly SC administrations of 1, 5 or 10 mg apraglutidewere scheduled on days 1, 8, 15, 22, 29 and 36.

FIG. 20A is a graph showing plasma apraglutide concentration (ng/mL) asa function of time (days) after the first dose of apraglutide.SC=subcutaneous; SD=standard deviation. Weekly SC administrations of 1,5 or 10 mg apraglutide were scheduled on day 1, 8, 15, 22, 29 and 36.Values below the limit of quantification (<1 ng/mL) were set to 0. Dataper dose level are presented as arithmetic means±SD on a linear scale.

FIG. 20B is a graph showing plasma apraglutide concentration (ng/mL) asa function of time (days) after the first dose of apraglutide.SC=subcutaneous; SD=standard deviation. Weekly SC administrations of 1,5 or 10 mg apraglutide were scheduled on day 1, 8, 15, 22, 29 and 36.Values below the limit of quantification (<1 ng/mL) were set to 0. Dataper dose level are presented as arithmetic means±SD on a log scale.

FIG. 21 is a graph showing individual Apraglutide C_(max) values andcorresponding citrulline R_(max) values (ug/mL) by dose level in Week 6.C_(max) Apraglutide (ng/mL); C_(max)=maximum concentration;R_(max)=maximum response. For the regression, the regression line, theequation, p-value of the slope and the R squared value are displayed.

FIG. 22 is a graph showing individual Apraglutide AUC_(tau) values andcorresponding citrulline R_(max) values (ug/mL) by dose level in Week 6.AUC_(tau)=area under the plasma concentration-time curve to the end ofthe treatment period of the corresponding dosing; R_(max)=maximumresponse. For the regression, the regression line, the equation, p-valueof the slope and the R squared value are displayed.

FIG. 23 is a correlation plot of individual apraglutide AUC_(tau) valuesand corresponding citrulline R_(max) values by dose level in Week 6. Theblack dot represents the assessment prior to the last dose and the lineconnects the subsequent assessments up to 2 weeks after the last dose.

FIG. 24 is a correlation plot of all apraglutide concentrations andcorresponding citrulline concentrations for each subject dosed with 1 mgin Week 6. The black dot represents the assessment prior to the lastdose and the line connects the subsequent assessments up to 2 weeksafter the last dose.

FIG. 25 is a correlation plot of all apraglutide concentrations andcorresponding citrulline concentrations for each subject dosed with 5 mgin Week 6. The black dot represents the assessment prior to the lastdose and the line connects the subsequent assessments up to 2 weeksafter the last dose.

FIG. 26A is a graph showing predicted plasma apraglutide concentrations(ng/mL) for a 70 kg individual receiving weekly subcutaneous apraglutide2.5, 5, or 10 mg.

FIG. 26B is a graph showing predicted plasma citrulline concentrations(ug/mL) for a 70 kg individual receiving weekly subcutaneous apraglutide2.5, 5, or 10 mg.

FIG. 27 is a graph showing individual and mean changes from baseline topost treatment in concentrations of plasma L-citrulline. Mean changefrom baseline was analyzed using a paired t-test. *p<0.05 Solid blackline represents mean change, colored lines represent individualpatients. Dotted line=SBS-II (intestinal insufficiency), solidline=SBS-IF (intestinal failure). A denotes the mean change frombaseline.

FIG. 28 is a graph showing citrulline plasma concentrations (ug/mL)following three weekly doses of apraglutide. Mean±standard error of themean (SEM) are shown.

FIG. 29 is a graph showing plasma apraglutide concentration (ng/mL) as afunction of time (hours) after the single subcutaneous 5 mg dose ofapraglutide.

DETAILED DESCRIPTION

Short bowel syndrome (SBS) is a disabling malabsorptive disorder causedby extensive surgical bowel resection. Patients with extensiveintestinal resections may suffer from disturbances in thegastrointestinal neuroendocrine feedback that regulate fluid andnutrient absorption. Disturbances include an impaired postprandialsecretion of GLP-2, normally produced by L-cells in the terminal ileumand colon. Lack of GLP-2 might result in an accelerated gastrointestinalemptying, gastrointestinal hypersecretion, diminished intestinal bloodflow, disturbed immunological and barrier function, and impaired mucosalgrowth. The consequent lack of intestinal adaptation contribute to thepathophysiological features of SBS, including frequent diarrheas orstoma emptyings, malnutrition, dehydration, electrolyte imbalances andweight loss. SBS is also associated with significant morbidity andmortality, and an impaired quality of life. In patients with SBS andintestinal failure (SBS-IF), parenteral support (PS), comprising anycombination of intravenous fluids and/or parenteral nutrition, isrequired to maintain health and/or growth.

Long-term PS provision may result in serious complications such ascatheter-related blood stream infections (CRBSI) and intestinalfailure-associated liver disease (IFALD). In contrast to patients withSBS-IF, patients with SBS intestinal insufficiency (SBS-II) managewithout PS due to their ability to compensate for their malabsorption byhyperphagia, metabolic adjustments and/or by pharmacological treatments.However, patients with SBS-II may be at risk of fluid and electrolyteimbalances which necessitate repeated hospital admissions for PSadministration. Collectively, patients with SBS have an impaired qualityof life, significant morbidity and mortality, and are health careexpensive. Globally, SBS-IF is a neglected organ failure with limitedtreatment possibilities. Thus, identifying new treatments could improvedisease awareness, morbidity and mortality, alleviate debilitatingsymptoms, and reduce patient treatment burden.

After surgical resection, the remnant bowel may undergo structural andfunctional changes to increase its absorptive capacity (commonlyreferred to as intestinal adaptation). The secretion of neuroendocrinepeptides throughout the gastrointestinal tract contribute to thisadaptation. The pathophysiological traits of SBS are often caused bydisturbance in the neuroendocrine feedback mechanisms and lack ofintestinal adaptation. This includes an impaired postprandial secretionof glucagon-like peptide-2 (GLP-2) which is produced by intestinalL-cells predominantly located in the terminal ileum and proximal colon.

Native GLP-2 has a short circulating half-life due to cleavage bydipeptidyl peptidase-IV (DPP4). Apraglutide is a next generationsynthetically manufactured GLP-2 analogue with a molecular structuredesigned to provide long-lasting constant exposure and an increasedhalf-life to at least 30 hours as compared with human GLP-2 and otherGLP-2 analogues. Apraglutide differs from human GLP-2 by four amino acidsubstitutions and was identified through chemistry structure-activityrelationship studies of lipophilic amino acid substitutions in positions11 and 16 of [Gly2] hGLP-2 (1-33). In animal models, apraglutidepromoted increase in intestinal length and weight, villus height andcrypt depth. Pharmacokinetic (PK) and pharmacodynamic studies in animalshave suggested that apraglutide may have a low clearance, longelimination half-life and a high plasma protein binding compared withother GLP-2 analogues. Therefore, apraglutide may be a candidate for aonce weekly dosing regimen. Apraglutide treatment can potentially helppatients to regain enteral autonomy or reduce PS requirements, improvesymptoms of malabsorption, alleviate organ failures secondary to II andIF, and prevent patients with SBS-II from deteriorating into a situationof intermittent or chronic IF.

As used herein, the term “amino acid” includes both naturally occurringamino acids and non-naturally occurring amino acids. Unless otherwisestated, an amino acid is an L-amino acid. Unless otherwise stated, aminoacid sequences are presented from the N-terminus to the C-terminus.

As used herein, the term “control,” when used alone or in reference to aquantity or level, may refer to the level observed in a subject beforeadministration of a treatment (e.g., an effective dose of a GLP-2agonist), a level observed in a control subject or a population ofsubjects (including historically observed levels). When used inreference to a subject, a “control” may refer to the same subject beforereceiving a given treatment (e.g., an effective dose of a GLP-2agonist), a similar subject who is not receiving any treatment for agiven condition, or a similar subject who is receiving a treatment(e.g., surgery, wound care, and/or nutritional support) that does notcomprise the given treatment (e.g., administration of an effective doseof a GLP-2 agonist).

As used herein, the term “GLP-2 agonist” refers collectively to ananalog of a naturally occurring GLP-2 in a vertebrate, which elicitssimilar or comparable activity to the naturally occurring GLP-2, but isstructurally altered, relative to a given vertebrate GLP-2, by at leastone amino acid addition, deletion, substitution, modification, and/or byincorporation of one or more amino acid(s) with a blocking group. Suchagonists preferably have an amino acid sequence at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% identical to that of either GLP-2 or a fragment ofGLP-2 having the same number of amino acid residues.

As used herein, the terms “patient” and “subject” are usedinterchangeably and refer to any subject for whom therapy is desired. Insome embodiments, the subject is a mammal, e.g., a human or a non-humanmammal. In some embodiments, the subject is a human being.

As used herein, the term “treatment” (also “treat” or “treating”) refersto any administration of a therapeutic entity (e.g., a therapeuticcompound or composition as described herein) that partially orcompletely reduces the need for parenteral support.

As used herein, the term “purity” is used to refer to purity asdetermined by chromatographic methods, more specifically by UltraPerformance Liquid Chromatography (UPLC) methods and/or High PerformanceLiquid Chromatography (HPLC) methods.

As used herein, the term “equivalent” of a substance (e.g. oxyma) isused to refer to a molar ratio of the substance, more specifically thenumber of moles of the substance that is to be reacted with one mole ofanother substance.

Any one of the embodiments and/or aspects described herein can becombined with any other embodiment and/or aspect described herein, andany number of embodiments and/or aspects can be combined.

GLP-2 agonists that can be used in methods of the present inventioninclude, for example, those disclosed in International PatentApplication No. WO2006/117565 and U.S. Pat. No. 8,589,918, the entirecontents of each which are herein incorporated by reference. TheGLP-agonists apraglutide and related compounds (see, e.g., U.S. Pat. No.8,589,918) have superior pharmacokinetic properties and are preferredfor use in the present methods.

SEQ ID NO: Amino acid sequence (GLP2) SEQ ID NO: 1His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH (Apraglutide) SEQ ID NO: 2His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Nle-D-Phe-Thr-Ile-Leu-Asp-Leu-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-NH₂

In preferred embodiments, the GLP-2 agonist is apraglutide, a peptidehaving the amino acid sequence of SEQ ID NO: 2, where Nle is norleucineand D-Phe is the D-amino acid phenylalanine.

Synthesis

Methods of preparing apraglutide have been described in the art andinclude solid phase peptide synthesis methods. See, for example, U.S.Pat. No. 8,580,918, which describes methods of preparing GLP-2 agonistsincluding apraglutide. As opposed to previous apraglutide synthesismethods (e.g. the methods disclosed in U.S. Pat. No. 8,580,918) thatwere only able to achieve a purity of 93%, the present disclosureprovides methods of making apraglutide that is substantially, e.g. ≥95%or ≥97%, pure, wherein the method still provides a high enough yield tobe used in commercial manufacturing processes. In addition, U.S. Pat.No. 8,580,918 discloses the ammonium salt of apraglutide. Disclosedherein is the synthesis of a preferred salt of apraglutide, the sodiumsalt.

The apraglutide production process is based on solid phase peptidesynthesis (SPPS) using Fmoc (9-fluorenylmethyloxycarbonyl) strategy. Theamino acid sequence is built stepwise from C-terminus by successivecycles of Fmoc deprotection and coupling of the next Fmoc amino acid.The novel synthesis method of apraglutide has a number of improvementsdescribed below. Fmoc-Rink-amide-MBHA (MethylBenzHydril Amine)-Resin isused, with the link between the peptide and the resin performed via aKnorr linker. After Fmoc deprotection the Piperidine/DMF (N,Ndimethylformamide) washes are done with use of Oxyma (Ethyl[hydroxyamino] cyanoacetate) in order to reduce levels of oxidationimpurities. Two DIC (Diisopropylcarbodiimide) additions are implementedin order to decrease the coupling time. After each amino acid couplingreaction, completeness is controlled by a semi-quantitative Kaiser testbased on revealing the unreacted amines. After assembly and the lastFmoc group deprotection, the peptide resin is washed with IPA(Isopropanol) and dried under vacuum. With use of novel synthesisprocess, the overall coupling time is reduced by around 25% compared toinitial condition, with no impact on crude peptide HPLC (HighPerformance Liquid Chromatography) purity and process yield.

After completion of peptide synthesis, cleavage of peptide from resin isdone by treatment with TFA (Trifluoroacetic acid)/H₂O/anisole mixture atroom temperature, under nitrogen. Cleaved peptide is separated fromresin by filtration and processed further in the improved downstreampurification steps to produce sodium salt of apraglutide with HPLCpurity ≥97%.

The improved version of apraglutide synthesis process (Process B;depicted in FIGS. 2A and 2B) comprises a primary purification by RP-HPLC(C18) chromatography in TFA-based mobile phases (H₂O/acetonitrile) to≥90% purity with pH of fractions adjusted using sodium bicarbonate(NaHCO₃), followed by secondary purification by RP-HPLC (C18) in NaHCO₃mobile phases (H₂O/acetonitrile) to ≥97% purity, and followed bydesalting/buffer exchange by RP-HPLC (C18) in sodium acetate(NaOAc)/H₂O/acetonitrile mobile phases. The product-containing fractionsare pooled and lyophilized to produce sodium salt of apraglutide with≥97% purity. The improved versions of the apraglutide synthesis processdescribed herein are able to provide highly pure apraglutide (e.g. ≥95%or ≥97% pure), while still maintaining yields that are suitable forlarge-scale commercial manufacture of apraglutide. As would beappreciated by the skilled artisan, methods of producing ultra-purecompounds typically suffer from low yields, making their use incommercial manufacturing infeasible. Surprisingly, the apraglutidesynthesis methods described herein not only provide highly pureapraglutide, but also exhibit yields that are suitable for use incommercial manufacturing contexts.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising:

a) performing solid phase peptide synthesis (SPPS) to synthesize theGLP-2 analog peptide on a resin;b) cleaving the synthesized GLP-2 analog peptide off the resin anddeprotecting the side chains of the synthesized GLP-2 analog peptide bytreating the resin with a solution comprising trifluoroacetic acid(TFA), water, and anisole;c) purifying the synthesized GLP-2 analog peptide using preparativereversed-phase high performance liquid chromatography (RP-HPLC);d) further purifying the product from step (c) using a secondpreparative RP-HPLC to produce a purified peptide solution (andoptionally a third preparative RP-HPLC where desalting and bufferexchange occurs);e) freeze-drying the purified peptide solution to produce a purifiedpeptide powder; andf) packaging the purified peptide powder under argon.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising: performing SPPS to synthesize the GLP-2 analogpeptide on a resin, wherein the resin is 4-Methylbenzhydrylamine (MBHA)resin.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising: performing SPPS to synthesize the GLP-2 analogpeptide on a resin, wherein the SPPS comprises washing the resin with asolution comprising DMF and oxyma.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising: performing SPPS to synthesize the GLP-2 analogpeptide on a resin, wherein SPPS comprises a coupling step performed bycontacting the resin with two amounts of a solution comprising DIC andoxyma, and wherein the amounts are contacted 30 minutes apart.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising: purifying the synthesized GLP-2 analog peptide bycontacting the peptide with a solution comprising water, acetonitrile(ACN), and ammonium hydroxide (NH₄OH) for the extraction of the crudepeptide from the cleaved peptide resin mixture and omitting thesubsequent acidification step.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising: purifying the synthesized GLP-2 analog peptide byRP-HPLC using a solution comprising 0.05% TFA, water, and acetonitrileas the eluent and adjusting the pH of the purified peptide fractions toabout pH 7.9 using 0.1% (acetic acid) AcOH in water.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising: purifying the synthesized GLP-2 analog peptide witha second purification by RP-HPLC using a solution comprising 0.05Msodium bicarbonate (NaHCO₃), water, and acetonitrile as the eluent.

The present disclosure provides a method of making a GLP-2 analogpeptide comprising: performing an HPLC purification using a 1.5 mMNaOAc/H₂O/ACN solution as the mobile phase.

In some aspects of the methods of the present disclosure, step (a)comprises:

i) preparing a resin on which the SPPS will be performed;ii) performing an initial Fmoc deprotection followed by a couplingreaction to add a first Fmoc-protected amino acid to the resin, therebyforming a protected peptide on the resin;iii) performing an Fmoc deprotection reaction followed by a couplingreaction to append at least one Fmoc-protected amino acid to theprotected peptide;iv) repeating step iii until the GLP-2 analog peptide is synthesized onthe resin, wherein a Fmoc-protected and side-chain protected GLP-2analog peptide is linked to the resin, and wherein the peptide comprisesthe amino acid sequence:L-histidyl-glycyl-L-aspartyl-glycyl-L-seryl-L-phenylalanyl-L-seryl-L-aspartyl-L-glutamyl-L-norleucyl-D-phenylalanyl-L-threonyl-Lisoleucyl-L-leucyl-L-aspartyl-L-leucyl-L-leucyl-L-alanyl-L-alanyl-L-arginyl-L-aspartyl-L-phenylalanyl-L-isoleucyl-L-asparaginyl-Ltryptophanyl-L-leucyl-L-isoleucyl-L-glutaminyl-L-threonyl-L-lysyl-L-isoleucyl-L-threonyl-Lasparticacid amide.v) performing an Fmoc deprotection reaction to produce a side-chainprotected GLP-2 analog peptide linked to the resin; andvi) drying the side-chain protected GLP-2 analog peptide linked to theresin.

In some aspects of the methods of the present disclosure, step (a)(i)comprises:

(a1) washing the resin with a solution comprising dimethylformamide(DMF) and N,N-Diisopropylethylamine (DIEA) at 5 mL of solution per gramof resin under an N₂ atmosphere;(b1) coupling a Rink amide linker to the resin in a solution comprising2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,

Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU), DIEA andHydroxybenzotriazole (HOBt) in DMF;

(c1) washing the product formed in step (b1) with DMF(d1) performing a reduction reaction by contacting the resin with asolution comprising acetic anhydride (Ac₂O) and DIEA in DMF; and(e1) washing the product formed in step (d1) with DMF.

In some aspects of the methods of the present disclosure, the resin isMethylbenzhydrylamine resin.

In some aspects of the methods of the present disclosure, the product ofsteps (a)(i)(a1)-(a)(i)(e1) is a Fmoc-Rink-amide-MBHA resin.

In some aspects of the methods of the present disclosure, step (c1)and/or step (e1) comprises washing the product three times at a ratio of5 milliliters of DMF to each gram of resin.

In some aspects of the methods of the present disclosure, a couplingtest may be performed between steps (d1) and (e1).

In some aspects of the methods of the present disclosure, a Kaiser testis performed to determine whether completion of the coupling wasachieved.

In some aspects of the methods of the present disclosure, the productupon completion of step (a)(i)(e1) is:Fmoc-His(Trt)-Gly-Asp(OtBu)-TmbGly-Ser(tBu)-Phe-Ser(tBu)-Asp(OtBu)-Glu(OtBu)-Nle-DPhe-Thr(tBu)-Ile-Leu-Asp(OtBu)-Leu-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Asn(Trt)-Trp(Boc)-Leu-Ile-Gln(Trt)-Thr(ψ^(Me,Me)pro)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Rink-amide-MBHA-Resin.

In some aspects of the methods of the present disclosure, performing anFmoc deprotection reaction followed by a coupling reaction comprises:

(a2) treating the resin with a solution comprising piperidine in DMF;(b2) washing the resin with DMF;(c2) contacting the resin with at least one Fmoc-protected amino acidand a solution comprising diisopropylcarbodiimide (DIC) and ethylcyanohydroxyiminoacetate (oxyma) in DMF, thereby coupling the at leastone Fmoc-protected amino acid; and(d2) washing the product formed in step (c2) with DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine in DMF is a 35% piperidine solution in DMF.

In some aspects of the methods of the present disclosure, treating theresin with the solution comprising piperidine in DMF is performed by a 3minute wash of the resin with the solution comprising piperidine in DMF,followed by a second 3 minute wash of the resin with the solutioncomprising piperidine in DMF, followed by a 10 minute wash of the resinwith the solution comprising piperidine in DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine in DMF is a 20% piperidine solution in DMF.

In some aspects of the methods of the present disclosure, the treatmentof the resin with the solution comprising piperidine in DMF is performedby a 15 minute wash of the resin with the solution comprising piperidinein DMF, followed by a second 15 minute wash of the resin with thesolution comprising piperidine in DMF.

In some aspects of the methods of the present disclosure, the washes areperformed at a ratio of 5 milliliters of solution comprising piperidinein DMF to each gram of resin.

In some aspects of the methods of the present disclosure, washing theresin in step (b2) comprises performing a first DMF wash at a ratio of15 milliliters of DMF to each gram of resin, followed by a second DMFwash at a ratio of 5 milliliters of DMF to each gram of resin.

In some aspects of the methods of the present disclosure, washing theresin in step (b2) comprises:

i) washing the resin with DMF; andii) washing the resin with a solution comprising DMF and oxyma.

In some aspects of the methods of the present disclosure, the solutioncomprising DMF and oxyma comprises 2 equivalents of oxyma.

In some aspects of the methods of the present disclosure, performing anFmoc deprotection reaction of residue Asp³ comprises treating the resinwith a solution comprising piperidine and Oxyma in DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine and Oxyma in DMF is a 10% piperidine and 2% Oxymasolution in DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine and Oxyma in DMF is a 5% piperidine and 1% Oxymasolution in DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine and Oxyma in DMF is a 15% piperidine and 3% Oxymasolution in DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine and Oxyma in DMF is a 20% piperidine and 4% Oxymasolution in DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine and Oxyma in DMF is a 25% piperidine and 5% Oxymasolution in DMF.

In some aspects of the methods of the present disclosure, the solutioncomprising piperidine and Oxyma in DMF is a 30% piperidine and 6% Oxymasolution in DMF.

In some aspects of the methods of the present disclosure, the treatmentof the resin with the solution comprising piperidine and Oxyma in DMF isperformed by a 15 minute wash of the resin with the solution comprisingpiperidine and Oxyma in DMF, followed by a second 30 minute wash of theresin with the solution comprising piperidine and Oxyma in DMF.

In some aspects of the methods of the present disclosure, step (c2)comprises:

i) contacting the resin with at least one Fmoc-protected amino acid anda solution comprising DIC and oxyma in DMF; andii) contacting the resin with a second amount of a solution comprisingDIC.

In some aspects of the methods of the present disclosure, the resin iscontacted with the second amount of a solution comprising DIC for about10, 15, 20, 25, 30, 35, 40, 45, or 50 minutes after contacting the resinwith the at least one Fmoc-protected amino acid.

In some aspects, the at least one protected amino acid is a di-peptidecomprising Boc-His(Trt)-Gly-OH.

In some aspects, the at least one protected amino acid is a di-peptidecomprising Fmoc-His(Trt)-Gly-OH.

In some aspects, the dipeptide is preactivated for 10 minutes, 30minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, or 4 hours beforecoupling the dipeptide.

In some aspects, the dipeptide is preactivated at 5° C.±2° C., 10° C.±2°C., 15° C.±2° C., 20° C.±2° C., 25° C.±2° C., 30° C.±2° C., or 35° C.±2°C. before coupling the dipeptide.

In some aspects, the dipeptide is preactivated in a solution comprisingBoc-His (Trt)-Gly-OH/Oxyma/DIC 2.5 mmol/2.5 mmol/2.5 mmol in 7 mL DMFbefore coupling the dipeptide.

In some aspects of the methods of the present disclosure, step (c2)comprises:

i) contacting the resin with at least one Fmoc-protected amino acid anda solution comprising DIC and oxyma in DMF; andii) contacting the resin with a second amount of a solution comprisingDIC and oxyma.

In some aspects of the methods of the present disclosure, the secondamount of a solution comprising DIC and oxyma is contacted with theresin about 30 minutes after contacting the resin with the at least oneFmoc-protected amino acid.

In some aspects of the methods of the present disclosure, in step (c2),the at least one Fmoc-protected amino acid is provided at aconcentration of 1, 2, 3, 4, or 5 equivalents.

In some aspects of the methods of the present disclosure, in step (c2),the DIC is provided at a concentration of 1, 2, 3, 4, or 5 equivalentsand the oxyma is provided at a concentration of 1, 2, 3, 4, or 5equivalents.

In some aspects of the methods of the present disclosure, in step (c2),the at least one Fmoc-protected amino acid isFmoc-Gln(Trt)-Thr(ψ^(Me,Me)pro)-OH.

In some aspects of the methods of the present disclosure, in step (c2),Fmoc-Gln(Trt)-Thr(ψ^(Me,Me)pro)-OH is provided at a concentration of 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 equivalents.

In some aspects of the methods of the present disclosure, the methodfurther comprises between step (b2) and step (c2), performing a test forresidual piperidine by measuring the amount of piperidine.

In some aspects of the methods of the present disclosure, if the amountof piperidine measured is greater than 10, 100, 200, 300, 400, 500, 600,700, 800, 900, or 1000 ppm, the resin is washed again with DMF and/or asolution comprising DMF and oxyma.

In some aspects of the methods of the present disclosure, a couplingtest is performed between step (c) and step (d).

In some aspects of the methods of the present disclosure, the couplingtest is a ninhydrin assay.

In some aspects of the methods of the present disclosure, step a(v)comprises:

(a3) treating the resin with a solution comprising piperidine in DMF;(b3) washing the resin with DMF;(c3) washing the resin with isopropanol.

In some aspects of the methods of the present disclosure, washing theresin in step (b3) comprises:

i) washing the resin with DMF; andii) washing the resin with a solution comprising DMF and oxyma.

In some aspects of the methods of the present disclosure, washing theresin with isopropanol in step (c3) comprises washing the resin fivetimes with isopropanol, wherein each wash is performed at a ratio of 5milliliters of isopropanol for each gram of resin.

In some aspects of the methods of the present disclosure, the productupon completion of step (a)(v) is a protected peptide on resincomprising:H-His(Trt)-Gly-Asp(OtBu)-TmbGly-Ser(tBu)-Phe-Ser(tBu)-Asp(OtBu)-Glu(OtBu)-Nle-DPhe-Thr(tBu)-Ile-Leu-Asp(OtBu)-Leu-Leu-Ala-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Ile-Asn(Trt)-Trp(Boc)-Leu-Ile-Gln(Trt)-Thr(ψ^(Me,Me)pro)-Lys(Boc)-Ile-Thr(tBu)-Asp(OtBu)-Rink-amide-MBHA-Resin.

In some aspects of the methods of the present disclosure, the solutioncomprising TFA, water and anisole is a solution comprising TFA, waterand anisole at a ratio of 95:2.5:2.5, TFA:water:anisole.

In some aspects of the methods of the present disclosure, the solutioncomprising TFA, water and anisole is a solution comprising TFA, waterand anisole at a ratio of 90:5:5, TFA:water:anisole.

In some aspects of the methods of the present disclosure, the solutioncomprising TFA, water and anisole is a solution comprising TFA, waterand anisole at a ratio of 80:10:10, TFA:water:anisole.

In some aspects of the methods of the present disclosure, the solutioncomprising TFA, water and anisole is a solution comprising TFA, waterand anisole at a ratio of 70:15:15, TFA:water:anisole.

In some aspects of the methods of the present disclosure, step (b)further comprises precipitating and washing the cleaved and deprotectedGLP-2 analog peptide using tert-butyl methyl ether (MTBE).

In some aspects of the methods of the present disclosure, the productupon completion of step (b) is:H-His-Gly-Asp-Gly-Ser⁵-Phe-Ser-Asp-Glu-NLe¹⁰-DPhe-Thr-Ile-Leu-Asp¹⁵-Leu-Leu-Ala-Ala-Arg²⁰-Asp-Phe-Ile-Asn-Trp²⁵-Leu-Ile-Gln-Thr-Lys³⁰-Ile-Thr-Asp-NH₂.

In some aspects of the methods of the present disclosure, step (c)comprises:

(i) contacting the product of step (b) with a solution comprising water,acetonitrile and NH₄OH;(ii) purifying the synthesized GLP-2 analog peptide using preparativereversed-phase high performance liquid chromatography (RP-HPLC).

In some aspects, the solution of step (c) (i) comprising water andacetonitrile comprises a mixture of H₂O/ACN in an 80:20 ratio in ammoniabuffer.

In some aspects, the solution of step (c) (i) comprising water andacetonitrile comprises a mixture of H₂O/ACN in a 70:30 ratio in ammoniabuffer.

In some aspects, the solution of step (c) (i) comprising water andacetonitrile comprises a mixture of H₂O/ACN in a 90:10 ratio, 80:20ratio, 70:30 ratio, 60:40 ratio, 50:50 ratio, or 40:60 ratio in ammoniabuffer.

In some aspects, the solution of step (c) (i) comprising water andacetonitrile is adjusted to target pH 7, pH 8, pH 9, pH 10, or pH 11.

In some aspects, the solution of step (c) (i) comprising water andacetonitrile is adjusted to target pH≥7.

In some aspects, the solution of step (c) (i) comprising water andacetonitrile is adjusted to target pH 8.0±0.1.

In some aspects, the solution of step (c) (i) comprising water andacetonitrile is adjusted to target pH 8.0±0.1, ±0.2, ±0.3, ±0.4, ±0.5,±0.6, ±0.7, ±0.8, or ±0.9.

In some aspects, the solution of step (c) (i) comprising water andacetonitrile is adjusted to about pH 10.

In some aspects of the methods of the present disclosure, the pH isadjusted using 25% acetic acid.

In some aspects of the methods of the present disclosure, the pH isadjusted using 25% NH₄OH in H₂O.

In some aspects, the solution of step (c) (i) is maintained at 0° C.,10° C., 20° C., 30° C., 40° C., 50° C., 60° C., 70° C., or 80° C.

In some aspects, the solution of step (c) (i) is maintained at 50° C.

In some aspects, the solution of step (c) (i) is maintained at RoomTemperature ° C.

In some aspects, the solution of step (c) (i) is maintained for 10minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 55 minutes, 60minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 90 minutes, 100minutes, 110 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 20hours, 24 hours, 30 hours, 40 hours, 50 hours, 60 hours, or 72 hours ata temperature disclosed herein.

In some aspects, the solution of step (c) (i) is maintained for 65minutes at a temperature disclosed herein.

In some aspects, the solution of step (c) (i) is maintained for 24 hoursat a temperature disclosed herein.

In some aspects, the solution of step (c) (i) is maintained for 65minutes at 50° C.

In some aspects, the solution of step (c) (i) is maintained for 24 hoursat Room Temperature ° C.

In some aspects of the methods of the present disclosure, the RP-HPLC isperformed using a C18 column.

In some aspects of the methods of the present disclosure, the RP-HPLC isperformed using a solution comprising NaHCO₃, water and acetonitrile asthe eluent.

In some aspects of the methods of the present disclosure, the solutioncomprising NaHCO₃, water and acetonitrile comprises 0.01M, 0.05M, 0.1M,0.2M, 0.3M, 0.4M, or 0.5M NaHCO₃.

In some aspects of the methods of the present disclosure, the RP-HPLC isperformed using a solution comprising TFA, water and acetonitrile as theeluent.

In some aspects of the methods of the present disclosure, the solutioncomprising TFA, water and acetonitrile comprises 0.01%, 0.05%, 0.1%,0.2%, 0.3%, 0.4%, or 0.5% TFA.

In some aspects of the methods of the present disclosure, the RP-HPLCcomprises adjusting the pH of purified peptide fractions.

In some aspects of the methods of the present disclosure, in step (d),the synthesized GLP-2 analog peptide is converted to its sodium cationform.

In some aspects of the methods of the present disclosure, step (d)comprises adjusting the pH of the purified peptide solution to about pH7.9 using 0.01%, 0.05%, 0.1%, 0.2%, 0.5%, 1%, or 2% AcOH in water.

In some aspects of the methods of the present disclosure, step (d)comprises adjusting the pH of the purified peptide solution to about pH7.9 using 0.1% AcOH in water.

In some aspects of the methods of the present disclosure, the productfrom step (c) is purified using HPLC, producing a further purifiedpeptide solution.

In some aspects of the methods of the present disclosure, the HPLC isperformed using a solution comprising AcOH, water and acetonitrile.

In some aspects of the methods of the present disclosure, the solutioncomprising AcOH, water and acetonitrile is a 0.01%, 0.05%, 0.1%, 0.5%,1%, 2%, 3%, 4%, or 5% AcOH solution.

In some aspects of the methods of the present disclosure, the HPLC isperformed using a solution comprising sodium acetate (NaOAc), water andacetonitrile.

In some aspects of the methods of the present disclosure, the solutioncomprising NaOAc, water and acetonitrile is a 0.1 mM, 0.5 mM, 1 mM, 1.5mM, 2 mM, 2.5 mM, or 3 mM NaOAc solution.

In some aspects of the methods of the present disclosure, following HPLCpurification, the pH of the further purified peptide solution isadjusted to about pH 7.9 using 0.1% AcOH in water.

In some aspects of the methods of the present disclosure, following HPLCpurification, the pH of the further purified peptide solution isadjusted to about pH 5, about pH 6, about pH 6.5, about pH 7, about pH7.5, about pH 7.9, about pH 8, about pH 8.5, or about pH 9 using 0.1%AcOH in water.

In some aspects, the starting materials presented in Table 1 can be usedin the methods of the present disclosure. In some aspects, the startingmaterials presented in Table 1 can have a purity that is presented inthe “Purity” column of Table 1:

TABLE 1 Abbreviation Material Purity Fmoc-Rink 4-[(2,4- ≥97%Amide-Linker Dimethoxyphenyl) (Fmoc- amino) methyl]- phenoxyacetic acidFmoc-Ala-OH, H₂O N^(α)-Fmoc-alanine ≥98% monohydrate ≤0.2% D-enantiomerFmoc-Arg(Pbf)-OH N^(α)-Fmoc-N^(ω)- ≥98% (2,2,4,6,7- ≤0.2% D-enantiomerpentamethyldihydroben zofuran-5- sulfonyl)- arginine Fmoc-Asn(Trt)-OHN^(α)-Fmoc-N^(γ)-trityl- ≥99% asparagine ≤0.2% D-enantiomerFmoc-Asp(OtBu)-OH N^(α)-Fmoc-aspartic acid ≥98% β-tertbutyl ester ≤0.2%D-enantiomer Fmoc-Glu(OtBu)- N^(α)-Fmoc-glutamic acid ≥98% OH•H₂Oγ-tertbutyl ester ≤0.2% D-enantiomer Fmoc-Gly-OH N^(α)-Fmoc-glycine ≥99%Fmoc-Gly(Tmb)-OH N^(α)-Fmoc-N^(α)-(2,4,6- ≥98% trimethox benzyl)glycineFmoc-His(Trt)-OH N^(α)-Fmoc-N^(τ)-histidine ≥98% ≤0.2% D-enantiomerFmoc-Ile-OH N^(α)-Fmoc-isoleucine ≥98% ≤0.1% Fmoc-D-Ile-OH ≤0.1%Fmoc-L-Allo-Ile- OH ≤0.1% Fmoc-D-Allo-Ile- OH Fmoc-Leu-OHN^(α)-Fmoc-leucine ≥98% ≤0.2% D-enantiomer Fmoc-Lys(Boc)-OHN^(α)-Fmoc-N^(ε)-tert- ≥98% butoxycarbonyl- lysine ≤0.2% D-enantiomerFmoc-Nle-OH N^(α)-Fmoc-norleucine ≥98% ≤0.5% D-enantiomer Fmoc-Phe-OHN^(α)-Fmoc-phenylalanine ≥99% ≤0.2% D-enantiomer Fmoc-D-Phe-OHN^(α)-Fmoc-D- ≥98% phenylalanine ≤0.5% L-enantiomer Fmoc-Ser(tBu)-OHN^(α)-Fmoc-O-tert-butyl- ≥98% serine ≤0.2% D-enantiomer Fmoc-Thr(tBu)-OHN^(α)-Fmoc-O-tert-butyl- ≥98% threonine ≤0.1% Fmoc-D- Thr(tBu)-OH ≤0.1%Fmoc-L-Allo- Thr(tBu)-OH ≤0.1% Fmoc-D-Allo- Thr(tBu)-OH Fmoc-Trp(Boc)-OHN^(α)-Fmoc-N^(in)-tert- ≥98% butoxycarbonyl- ≤0.2% D-enantiomertryptophan Fmoc-Gln(Trt)- N^(α)-Fmoc-N^(δ)-trityl- ≥98%Thr(ψ^(Me, Me)Pro)-OH glutaminyl-2,2, 5- ≤0.5% D-enantiomertrimethyloxazolidine-4- carboxylic acid

In some aspects of the methods of the present disclosure, theapraglutide has a purity of no less than 95%.

In some aspects of the methods of the present disclosure, theapraglutide has a purity of no less than 97%.

In some aspects, the concentrations and/or presence of Des-Gly⁴apraglutide, Aspartimide³, Asp³³-OH, Des-Ser⁷ apraglutide, and [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] can be determined using RP-HPLC. Table 2ashows the purity and major impurities in two variants of the abovedescribed synthesis. Table 2b shows the purity and yield in threevariants of the apraglutide synthesis process described herein.

TABLE 2a Process A Process B ≥95% (FIG. 2A and 2B) ≥95% API API ≥97% APIpurity test purity ≥97% API Test specification result specification testresult Purity ≥95.0% 95.6% ≥97.0% 97.9% Sum of related ≤5.0%  4.4% ≤3.0%2.12% impurities Des-Gly⁴ & ≤1.0% 0.42% ≤1.0% 0.53% Aspartimide³ (twoco-eluting impurities) Asp³³ ≤1.0% 0.45% ≤1.0% 0.59% Des-Ser⁷ ≤1.0%0.13% ≤1.0% 0.27% Trp²⁵ 2-(2′4′6- trimethoxyphenyl) ≤2.0% 0.78% ≤1.0%<0.05%  D-Asp³ ≤2.0% 0.22% ≤1.0% Not detected API Content ≥80.0% 85.7%≥85.0% 91.2%

TABLE 2b Process A Process A Process A Process B Process B Process C Run#1 Run #2 Run #3 Run #1 Run #2 Run #1 Chromatographic 92.3% 91.5% 95.3%97.9% 96.3% 98.5% Purity Yield   15%   22%   17% 20.7% 20.5% 22.4%

In some embodiments, the purified peptide pool does not contain anyunspecified impurity at a concentration greater than 0.01%, 0.05%, 0.1%,0.5%, 1%, 1.5%, or 2%. In some embodiments, the purified peptide pooldoes not contain any unspecified impurity at a concentration greaterthan 1%.

In some embodiments, the purified peptide pool does not contain animpurity comprising Des-Gly⁴ GLP-2 analog at a concentration greaterthan 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%, 2.5%, 3%, 3.5%, 4%, or5%. In some embodiments, the purified peptide pool does not containDes-Gly⁴ GLP-2 analog at a concentration greater than 3%.

In some embodiments, the purified peptide pool does not contain animpurity comprising: Sum of Aspartimide³, Asp³³-OH and Des-Ser⁷-GLP-2analog impurities at a concentration greater than 0.01%, 0.05%, 0.1%,0.5%, 1%, 1.5%, or 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments,the purified peptide pool does not contain Sum of Aspartimide³, Asp³³-OHand Des-Ser⁷-GLP-2 analog impurities at a concentration greater than 2%.

In some embodiments, the purified peptide pool does not contain animpurity comprising: [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] GLP-2 analog ata concentration greater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%,2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the purified peptidepool does not contain [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] GLP-2 analogimpurity at a concentration greater than 2%.

In some embodiments, the purity of the GLP-2 analog peptide sodium saltas measured by RP-HPLC is greater than 95%, 97% or 99%. In someembodiments, the purity of purified peptide pool as measured by RP-HPLCis greater than 95%.

In some embodiments, the purity of the GLP-2 analog peptide sodium saltdoes not contain any unspecified impurity at a concentration greaterthan 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%. In some embodiments, theGLP-2 analog peptide sodium salt does not contain any unspecifiedimpurity at a concentration greater than 1%.

In some embodiments, the GLP-2 analog peptide sodium salt does notcontain an impurity comprising Des-Gly⁴ GLP-2 analog at a concentrationgreater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,or 5%. In some embodiments, the GLP-2 analog peptide sodium salt doesnot contain Des-Gly⁴ GLP-2 analog impurity at a concentration greaterthan 3%.

In some embodiments, the GLP-2 analog peptide sodium salt does notcontain an impurity comprising: sum of Aspartimide³, Asp³³-OH andDes-Ser⁷-GLP-2 analog impurities at a concentration greater than 0.01%,0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In someembodiments, the GLP-2 analog peptide sodium salt does not contain thesum of Aspartimide³, Asp³³-OH and Des-Ser⁷-GLP-2 analog impurities at aconcentration greater than 2%.

In some embodiments, the GLP-2 analog peptide sodium salt does notcontain an impurity comprising: [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)]GLP-2 analog at a concentration greater than 0.01%, 0.05%, 0.1%, 0.5%,1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2analog peptide sodium salt does not contain [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] GLP-2 analog impurity at a concentrationgreater than 2%.

In some aspects of the methods of the present disclosure, the method canfurther comprise an in-process control after the conversion of thepeptide to the sodium salt form, wherein the in-process control isperformed by RP-HPLC and the acceptance criteria is that the peptide hasa purity of ≥95.0% and no more than the following impurities: Des-Gly⁴apraglutide is ≤3.0%, Sum of Aspartimide³, Asp³³-OH and Des-Ser⁷apraglutide is ≤2.0%, [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] apraglutide is≤2.0% and any other unspecified impurity is ≤1.0%.

Without wishing to be bound by theory, in some embodiments, the use ofFmoc-Gln(Trt)-Thr(ψ^(Me,Me)Pro)-OH and Fmoc-Tmb-Gly-OH during Fmocdeprotection and coupling cycles improves coupling efficiency andreduces aspartimide formation.

Without wishing to be bound by theory, in some embodiments, the use ofoxyma as the coupling additive during Fmoc deprotection and couplingcycles minimizes formation of oxidation and [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] GLP-2 analog impurities.

Without wishing to be bound by theory, in some embodiments, the use ofACN for the mobile phase composition during sodium salt conversion bypreparative RP-HPLC improves GLP-2 analog peptide composition sodiumsalt conversion. In some embodiments, and the removal of a titrationstep during sodium salt conversion by preparative RP-HPLC improves GLP-2analog peptide composition sodium salt conversion. In some embodiments,the introduction of pH adjustment during sodium salt conversion bypreparative RP-HPLC improves GLP-2 analog peptide composition sodiumsalt conversion.

The Des-Gly⁴ GLP-2 analog impurity comprises Gly⁴ missing in thepeptide. The Aspartimide³ GLP-2 analog impurity comprises Aspartimideformation during Asp³ coupling. The Asp³³-OH GLP-2 analog impuritycomprises C-terminal amide hydrolysis of the peptide. The Des-Ser⁷ GLP-2analog impurity comprises Ser⁷ missing in the peptide. The [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] GLP-2 analog impurity comprisesFmoc-Gly(Tmb)-OH formation during the cleavage process.

In some embodiments, the GLP-2 analog peptide composition does notcontain any individual impurity at a concentration greater than 0.01%,0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4%. In someembodiments, the GLP-2 analog peptide composition does not contain anyindividual impurity at a concentration greater than 1.5%.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising Des-Gly⁴ GLP-2 analog at a concentrationgreater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,or 5%. In some embodiments, the GLP-2 analog peptide composition doesnot contain a Des-Gly⁴ GLP-2 analog impurity at a concentration greaterthan 3%.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising β-Asp³ GLP-2 analog at a concentrationgreater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,or 5%. In some embodiments, the GLP-2 analog peptide composition doesnot contain a β-Asp³ GLP-2 analog impurity at a concentration greaterthan 1.5%. In some embodiments, the GLP-2 analog peptide compositiondoes not contain a β-Asp³ GLP-2 analog impurity at a concentrationgreater than 1%.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising D-His GLP-2 analog at a concentrationgreater than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,or 5%. In some embodiments, the GLP-2 analog peptide composition doesnot contain a D-His GLP-2 analog impurity at a concentration greaterthan 1.5%. In some embodiments, the GLP-2 analog peptide compositiondoes not contain a D-His GLP-2 analog impurity at a concentrationgreater than 1%.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising the sum of Aspartimide³, Asp³³-OH andDes-Ser⁷-GLP-2 analog impurities at a concentration greater than 0.01%,0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In someembodiments, the GLP-2 analog peptide composition does not contain thesum of Aspartimide³, Asp³³-OH and Des-Ser⁷-GLP-2 analog impurities at aconcentration greater than 2%.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising: [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)]GLP-2 analog at a concentration greater than 0.01%, 0.05%, 0.1%, 0.5%,1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 5%. In some embodiments, the GLP-2analog peptide composition does not contain [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] GLP-2 analog impurity at a concentrationgreater than 2%.

In some embodiments, the GLP-2 analog peptide does not contain animpurity comprising: Des-Gly⁴ GLP-2 analog, Aspartimide³ GLP-2 analog,Asp³³-OH GLP-2 analog, Des-Ser⁷ GLP-2 analog, or [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] GLP-2 analog at a concentration greater than0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2%, 2.5%, 3%, 3.5%, 4%, or 5%.

In some embodiments, the GLP-2 analog peptide does not contain animpurity comprising: Des-Gly⁴ GLP-2 analog peptide, Aspartimide³ GLP-2analog peptide, Asp³³-OH GLP-2 analog peptide, Des-Ser⁷ GLP-2 analogpeptide, or [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] GLP-2 analog peptide ata concentration greater than 0.5%.

In some embodiments, the GLP-2 analog peptide composition does notcontain a total amount of impurities at a concentration greater than 1%,3%, 5%, or 7%. In some embodiments, the GLP-2 analog peptide compositiondoes not contain a total amount of impurities at a concentration greaterthan 5%.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising acetate at a concentration greater than1%, 2%, 3%, 4%, or 5%. In some embodiments, the GLP-2 analog peptidecomposition does not contain acetate at a concentration greater than 5%.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising trifluoroacetic acid at a concentrationgreater than 0.01%, 0.03%, 0.05%, 0.07%, 0.1%, 0.2%, 0.5%, or 1%. Insome embodiments, the GLP-2 analog peptide composition does not containtrifluoroacetic acid at a concentration greater than 0.1%.

In some embodiments, the GLP-2 analog peptide composition contains animpurity comprising acetonitrile at less than 5,000, 4,000, 3,000,2,000, 1,000, 500, 400, 300, 200, or 100 ppm. In some embodiments, theGLP-2 analog peptide composition contains acetonitrile at less than 450ppm.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising bacterial endotoxins at a concentrationgreater than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg. In someembodiments, the GLP-2 analog peptide composition does not containbacterial endotoxins at a concentration greater than 5.0 EU/mg.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising aerobic microbes at a concentrationgreater than 1, 10, 50, 100, 150, or 200 CFU/0.1 g. In some embodiments,the GLP-2 analog peptide composition does not contain aerobic microbesat a concentration greater than 100 CFU/0.1 g.

In some embodiments, the GLP-2 analog peptide composition does notcontain an impurity comprising yeasts and mold at a combinedconcentration greater than 0.1, 1, 5, 10, 15, or 20 CFU/0.1 g. In someembodiments, the GLP-2 analog peptide composition does not containyeasts and mold at a combined concentration greater than 10 CFU/0.1 g.

In some embodiments, the GLP-2 analog peptide composition does notcontain impurities comprising acetate, trifluoroacetic acid,acetonitrile, bacterial endotoxins, aerobic microbes, yeasts, mold, orany combination thereof.

After synthesis, the apraglutide active pharmaceutical ingredient(“API”) is lyophilized and thereafter formulated into a drug product.

Formulation

GLP-2 agonists of the present disclosure can use used alone as apharmaceutical or in a pharmaceutical composition comprising one or moreGLP-2 agonists as the active ingredient(s) and a pharmaceuticallyacceptable adjuvant, excipient, diluent, or carrier. Pharmaceuticalcompositions may also comprise other active ingredients.

Suitable pharmaceutically acceptable carriers include those typicallyused with peptide-based drugs. See, e.g., “Remington: The Science andPractice of Pharmacy,” 22nd ed., Pharmaceutical Press, Philadelphia,Pa., 2012, for general guidance on drug formulations. Non-limitingexamples of suitable excipients include glycine, L-histidine, mannitol,and any combination thereof.

A preferred formulation uses Glycine as a buffering agent. L-Histidineis used in this formulation as a physically stabilizing agent.L-Histidine also serves as a buffer, maintaining the target pH. Mannitolis used in this formulation as a bulking agent in the lyophilizationprocess step. Water for injection is the solvent of this formulation;the water for injection is removed during the lyophilization step.Sodium hydroxide is used for pH adjustment. pH is measured and may beadjusted to 8.30±0.10 with sodium hydroxide (NaOH) solution.

The present disclosure provides a robust and reproducible lyophilizationcycle for the manufacture of the drug product. Using the same containerclosure intended to be used in the manufacture of the clinical batch,two scaled down development batches were filled. The lyophilizationcycle was optimized for primary drying (primary drying cycle) to definethe process to be implemented in clinical manufacturing of the drugproduct. The optimized lyophilization cycle resulted in a drug productwith the expected quality attributes (i.e., assay, purity and impuritylevels, reconstitution time and moisture content) and was deemedappropriate for a clinical batch of the drug product. A stability studyconducted on the second scaled-down development batch demonstrated thedrug product remained unchanged at the intended storage temperature (2to 8° C.) for up to 3 months.

In some aspects, the composition of apraglutide for injection andreconstituted solution presented in Table 3 is an exemplary compositionthat may be produced using the methods of the present disclosure.

TABLE 3 Quantity Apraglutide for Reconstituted Name of InjectionSolution Ingredient Reference Function (mg/mL) (mg/mL) ApraglutideIn-house Drug substance 12.5^(a) 25 Glycine Ph. Eur/USP Buffering 1.883.75 Agent L-Histidine Ph. Eur/USP Stabilizing 3.88 7.75 Agent MannitolPh. Eur/USP Bulking Agent 57.5 115.0 Sodium Ph. Eur/USP pH adjustmentq.s. to — Hydroxide pH 8.3 Water for Ph. Eur/USP Solvent 1.25 ml —Injection (WFT)^(b) Nitrogen^(b) NF Inert Overlay — — Sterile Ph.Eur/USP Reconstitution — 0.5 mL Water for diluent Injection ^(a)Thequantity of drug substance to be used is calculate based on theapraglutide content in the corresponding drug substance batch.^(b)Components used during the manufacture of the drug product that donot appear in the final product.

In some embodiments, the apraglutide composition may include excipientscomprising glycine, L-histidine, mannitol, water for injection (WFI),sterile water for injection (sWFI), sodium hydroxide, sucrose, or anycombination thereof.

In one aspect the present disclosure provides a pharmaceuticalcomposition comprising apraglutide of the present disclosure, glycine,L-histidine and mannitol dissolved in water, wherein the concentrationof the apraglutide is 25 mg/mL, the concentration of glycine is 3.75mg/mL, the concentration of L-histidine is 7.75 mg/mL of water and theconcentration of mannitol is 115.0 mg/mL. In some aspects, the volume ofwater can be 0.5 mL. In some aspects, the preceding pharmaceuticalcomposition can further comprise sodium hydroxide in an amount such thatthe pH of the solution is about pH 8.3.

In some aspects, the osmolarity of a pharmaceutical composition of thepresent disclosure is between 290-780 mOsmol/kg. In some aspects, theosmolarity of a pharmaceutical composition of the present disclosure isabout 780 mOsmol/kg. In some aspects, the osmolarity of a pharmaceuticalcomposition of the present disclosure is about 780±160 mOsmol/kg.

The GLP-2 analog peptide composition for injection may be an asepticallymanufactured lyophilized powder for solution for injection. It can bepresented in a colorless glass vial suitable for a lyophilized sterileproduct, closed with a rubber stopper and sealed with an aluminum cap.Prior to administration, the GLP-2 analog peptide for injection may bedissolved in 0.5 mL of sterile Water for Injection (sWFI). Thereconstituted solution may be administered subcutaneously.

Administration

In some embodiments, GLP-2 agonists, e.g., apraglutide, are administeredparenterally, e.g. by injection. Formulations suitable for parenteraladministration include aqueous and non-aqueous, isotonic sterileinjection solutions, which can contain antioxidants, buffers,bacteriostatics, and solutes that render the formulation isotonic withthe blood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Liquid carriers, for injectablesolutions, include by way of example and without limitation water,saline, aqueous dextrose and glycols.

In some aspects, the apraglutide is delivered via a two chamber syringeor dual cartridge injector. One example of such a syringe is describedin PCT/EP2012/000787, the contents of which are incorporated herein byreference.

Dosing

Amounts that constitute an effective dose may depend on various factorssuch as the disease and clinical status of the patient (e.g. weight) andthe route of administration. Doses may be administered, e.g., twicedaily, once daily, twice a week, weekly, biweekly, once or twicemonthly, etc. Doses generally range from about 1 mg to about 10 mg perweek for a period of about 1 week to about 100 weeks. In someembodiments, the weekly dose is between about 1 mg and 10 mg. In someembodiments, subjects are dosed from between about 1 weeks to about 100weeks, about 1 weeks to about 80 weeks, about 1 weeks to about 60 weeks,about 1 weeks to about 48 weeks, about 2 weeks to about 24 weeks, about2 weeks to about 20 weeks, or about 2 weeks to about 16 weeks. In someembodiments, subjects are administered a dose about once a week. In someembodiments, subjects are administered a dose about once every two weeksor about twice a month. About once every two weeks or about twice amonth.

Apraglutide increases citrulline levels in a dose dependent manner.Citrulline is a marker of small bowel enterocyte mass. Dosing ofapraglutide at 1 mg, 5 mg and 10 mg induces long-lasting increases incitrulline concentration in patients.

In some embodiments, apraglutide is administered parenterally (e.g.,subcutaneously, intravenously, intramuscularly, or orally). In someembodiments, apraglutide is administered intravenously. In someembodiments, apraglutide is administered subcutaneously.

Due to the non-linear increase of exposures (AUC and C_(max)) as bodyweight decreases, it is desired to dose patients with body weight below50 kg with 2.5 mg to prevent high exposures. Patients of 50 kg or highermay receive 5 mg or higher doses.

Indications

In some embodiments, a subject who has been administered an effectivedose of a GLP-2 agonist needs less nutritional support relative to acontrol. For example, in some embodiments, a subject who has beenadministered an effective dose of GLP-2 agonist needs at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90% less nutritional supportrelative to a control. In one embodiment, a subject who has beenadministered an effective dose of GLP-2 agonist needs 100% lessnutritional support relative to a control (i.e., enteral autonomy).

In some embodiments, a subject who has been administered an effectivedose of a GLP-2 agonist needs total parenteral support for fewer daysrelative to a control. For example, in some embodiments, administrationof an effective dose of a GLP-2 agonist may reduce the length of timeduring which total parenteral support is needed by at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%relative to a control. In one embodiment, administration of an effectivedose of GLP-2 agonist reduces the length of time during which totalparenteral support is needed by 100% relative to a control (i.e.,enteral autonomy). In some embodiments, a subject who has beenadministered an effective dose of a GLP-2 agonist needs total parenteralsupport for less than 85 days per year, less than 80 days per year, lessthan 75 days per year, less than 70 days per year, less than 65 days peryear, less than 60 days per year, less than 55 days per year, less than50 days per year, less than 45 days per year, less than 40 days peryear, less than 35 days per year, less than 30 days per year, less than25 days per year, less than 20 days per year, less than 15 days peryear, less than 10 days per year, or less than 5 days per year.

In some embodiments, a subject who is administered an effective dose ofa GLP-2 agonist does not need total parenteral nutrition followingtreatment.

In some embodiments the patients receiving apraglutide include male andfemale subjects with short bowel syndrome associated intestinal failure(“SBS-IF”), receiving parenteral support, secondary to surgicalresection of the small intestine with either:

a. Colon-in-continuity (“CIC”) remaining and no stoma (small intestine<200 cm from duodeno-jejunal flexure, based on availablemedical/surgical records) with the latest intestinal resection being atleast 12 months prior to screening OR

b. Jejunostomy or ileostomy (<200 cm from duodeno-jejunal flexure, basedon available medical/surgical records) with the latest intestinalresection being at least 6 months prior to screening.

In some embodiments, administration of an effective dose of a GLP-2agonist results in reduced hospital length of stay relative to acontrol. For example, in some embodiments, administration of aneffective dose of a GLP-2 agonist may reduce the length of a hospitalstay by at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95% relative to a control. In some embodiments, a subjectwho has been administered an effective dose of a GLP-2 agonist ishospitalized for less than 50 days, less than 45 days, less than 40days, less than 35 days, less than 30 days, less than 25 days, less than20 days, less than 15 days, less than 10 days, or less than 5 days.

In some embodiments, administration of an effective dose of a GLP-2agonist to a population of subjects reduces the mortality rate relativeto a population of subjects who receive a standard of care treatment(e.g., surgical treatment, nutritional support, and wound care) thatdoes not comprise administration of an effective dose of a GLP-2agonist. For example, in some embodiments, the mortality rate is reducedto less than 15%, less than 12%, less than 10%, less than 8%, less than5%, less than 3%, less than 2%, or less than 1%. The SC injection willtypically be administered in the abdominal area or in the thigh. Theinjection site should be rotated such that an injection is administeredat least 5 cm away from where the last injection was administered.

It is believed that apraglutide can demonstrate clinical efficacy(reduction in PS volume) in SBS-IF subjects with either CIC or stomaafter 24 weeks of once a week dosing. Accordingly, in one embodiment asingle 2.5 mg dose (for subjects with body weight less than 50 kg atmost recent trial visit) or 5 mg dose (for subjects with body weight 50kg or more at most recent trial visit) of apraglutide or matchingplacebo will be administered by subcutaneous (“Sc”) injection onceweekly during a treatment period of 24 weeks (stoma) or 48 weeks (CIC).

Apraglutide has demonstrated superior effects on energy absorption withless frequent dosing (i.e., once weekly) compared to other GLP-2analogs. When dosed at 5 mg once weekly, apraglutide caused a 140.1%(±15.8) change from baseline increase in urinary input, a change inbaseline in wet weight absorption of 760.4 g/day (±236.1), and a changein baseline in energy absorption of 1,074 kj/day (±377).

Apraglutide promoted the increase of absolute urine volume output by anadjusted mean of 711 mL/day (95% CI 132 to 1,289; P=0.021) compared toplacebo, corresponding to a daily increase of 48% (95% CI 12 to 84;P=0.014), as shown in Table 9. Apraglutide promoted the increase ofabsolute urine volume output by an adjusted mean of 714 mL/day (95% CI490 to 939; P=0.002), corresponding to a daily increase of 49% (95% CI 4to 94; P=0.041), as shown in Table 10. Treatment with 10 mg apraglutidepromoted the increase of absolute urine volume output by an adjustedmean of 795 mL (95% CI 195 to 1,394; P=0.014) compared to placebo. Thecorresponding change in relative urine production was 34% (95% CI −4 to71; P=0.072).

Apraglutide increased urine sodium excretion compared to placebo by anadjusted mean of 56 mmol/day (95% CI −10 to 123; P=0.087), as shown inTable 9. 5 mg apraglutide increased urine sodium excretion compared toplacebo by an adjusted mean of 66 mmol/day (95% CI −69 to 201; P=0.171),as shown in Table 10. In the 10 mg dose group, absolute urine sodiumexcretion was increased by an adjusted mean of 88 mmol/day (95% CI 20 to156; P=0.017) compared to placebo, as shown in Table 9.

Apraglutide increased intestinal absorption of energy by 1,095 kJ/day(95% CI 196 to 1,994; P=0.024), as shown in Table 19.

By contrast, teduglutide, dosed once daily, caused a 139.3% change frombaseline increase in urinary input, a change in baseline in wet weightabsorption of 743 g/day (±119.25), and a change in baseline in energyabsorption of 792 kj/day (±570), as reported in Jeppesen, Gut, 54, pp.1224-1231 (2005).

Similarly, by contrast, glepaglutide, dosed once daily, caused a 111%(0.1 mg dose), 140% (1.0 mg dose) or 132% (10 mg dose) change frombaseline increase in urinary output, a change in baseline in wet weightabsorption of −211 g/day (0.1 mg dose), 650 g/day (1.0 mg dose) or 786g/day (10 mg dose), and a change in baseline in energy absorption of−377 kj/day (0.1 mg dose), 435 kj/day (1.0 mg dose) or 588 kj/day (10 mgdose), as reported in Maimi, Lancet Gastroenterol Hepatol, 4, pp.354-363 (2019).

Any use of growth hormone, glutamine or growth factors such as nativeGLP-2, GLP-1 or GLP-2, GLP-1 analogues other than the IMP underinvestigation should be discontinued for 12 month (CIC subjects) and 6months (stoma subjects) before administration of the apraglutide.

In some embodiments, administration of an effective dose of a GLP-2agonist to a population of subjects reduces the morbidity rate relativeto a population of subjects who receive a standard of care treatment(e.g., surgical treatment, nutritional support, and wound care) thatdoes not comprise administration of an effective dose of a GLP-2agonist. For example, in some embodiments, the morbidity rate is reducedto less than 85%, less than 80%, less than 75%, less than 70%, less than65%, less than 60%, less than 55%, less than 50%, less than 45%, lessthan 40%, less than 35%, less than 30%, less than 25%, less than 20%,less than 15%, or less than 10%.

In some embodiments, apraglutide is used for post-surgical enteralautonomy recovery or for treatment of enterocutaneous fistulas. In someembodiments, apraglutide is used for treatment of anastomotic leaks,functional intestinal failure, intestinal insufficiency, necrotizingenterocolitis, graft versus host disease, Crohn's disease or celiacdisease.

The present disclosure provides a method of treating short bowelsyndrome in a subject, the method comprising administering at least onetherapeutically effective amount of a GLP-2 analog peptide compositionof the present disclosure.

The present disclosure provides a GLP-2 analog peptide composition ofthe present disclosure for use in the treatment of short bowel syndromein a subject, wherein the GLP-2 analog peptide composition of thepresent disclosure is for administration to the subject in at least onetherapeutically effective amount.

The present disclosure provides a GLP-2 analog peptide composition ofthe present disclosure for the manufacture of a medicament for treatingof short bowel syndrome in a subject, wherein the GLP-2 analog peptidecomposition of the present disclosure is for administration to thesubject in at least one therapeutically effective amount.

The present disclosure provides a method for increasing the intestinalabsorption of wet weight in a subject with short bowel syndrome, themethod comprising administering to the subject at least onetherapeutically effective amount of a GLP-2 analog peptide compositionof the present disclosure. The present disclosure provides a GLP-2analog peptide composition of the present disclosure for use in a methodof increasing the intestinal absorption of wet weight in a subject withshort bowel syndrome, wherein the GLP-2 analog peptide composition ofthe present disclosure is for administration to the subject in at leastone therapeutically effective amount. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use inthe manufacture of a medicament for increasing the intestinal absorptionof wet weight in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount.

In some aspects, the increase in intestinal absorption of wet weight canbe at least about 100 g/day, or at least about 200 g/day, or at leastabout 300 g/day, or at least about 400 g/day, or at least about 500g/day, or at least about 600 g/day, or at least about 700 g/day, or atleast about 800 g/day, or at least about 900 g/day, or at least about1000 g/day.

The present disclosure provides a method for decreasing fecal output ina subject with short bowel syndrome, the method comprising administeringto the subject at least one therapeutically effective amount of a GLP-2analog peptide composition of the present disclosure. The presentdisclosure provides a method for decreasing stoma output in a subjectwith short bowel syndrome, the method comprising administering to thesubject at least one therapeutically effective amount of a GLP-2 analogpeptide composition of the present disclosure. The present disclosureprovides a GLP-2 analog peptide composition of the present disclosurefor use in a method of decreasing the fecal output in a subject withshort bowel syndrome, wherein the GLP-2 analog peptide composition ofthe present disclosure is for administration to the subject in at leastone therapeutically effective amount. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use inthe manufacture of a medicament for decreasing the fecal output in asubject with short bowel syndrome, wherein the GLP-2 analog peptidecomposition of the present disclosure is for administration to thesubject in at least one therapeutically effective amount.

In some aspects, the decrease in fecal output can be at least about 100g/day, or at least about 200 g/day, or at least about 300 g/day, or atleast about 400 g/day, or at least about 500 g/day, or at least about600 g/day, or at least about 700 g/day, or at least about 800 g/day, orat least about 900 g/day, or a least about 1000 g/day.

The present disclosure provides a method for increasing urine productionin a subject with short bowel syndrome, the method comprisingadministering to the subject at least one therapeutically effectiveamount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition ofthe present disclosure for use in a method of increasing the urineproduction in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount. The present disclosure provides a GLP-2 analog peptidecomposition of the present disclosure for use in the manufacture of amedicament for increasing the urine production in a subject with shortbowel syndrome, wherein the GLP-2 analog peptide composition of thepresent disclosure is for administration to the subject in at least onetherapeutically effective amount.

In some aspects, the increase in urine production can be at least about100 g/day, or at least about 200 g/day, or at least about 300 g/day, orat least about 400 g/day, or at least about 500 g/day, or at least about600 g/day, or at least about 700 g/day, or at least about 800 g/day, orat least about 900 g/day, or a least about 1000 g/day.

The present disclosure provides a method for increasing absorption ofsodium and/or potassium in a subject with short bowel syndrome, themethod comprising administering to the subject at least onetherapeutically effective amount of a GLP-2 analog peptide compositionof the present disclosure. The present disclosure provides a GLP-2analog peptide composition of the present disclosure for use in a methodof increasing the absorption of sodium and/or potassium in a subjectwith short bowel syndrome, wherein the GLP-2 analog peptide compositionof the present disclosure is for administration to the subject in atleast one therapeutically effective amount. The present disclosureprovides a GLP-2 analog peptide composition of the present disclosurefor use in the manufacture of a medicament for increasing the absorptionof sodium and/or potassium in a subject with short bowel syndrome,wherein the GLP-2 analog peptide composition of the present disclosureis for administration to the subject in at least one therapeuticallyeffective amount.

In some aspects, the increase in absorption of sodium and/or potassiumcan be at least about 5 mmol/day, or at least about 10 mmol/day, or atleast about 15 mmol/day, or at least about 20 mmol/day, or at leastabout 25 mmol/day, or at least about 30 mmol/day, or at least about 35mmol/day, or at least about 40 mmol/day, or at least about 45 mmol/day,or at least about 50 mmol/day.

The present disclosure provides a method for increasing urine sodiumand/or potassium excretion in a subject with short bowel syndrome, themethod comprising administering to the subject at least onetherapeutically effective amount of a GLP-2 analog peptide compositionof the present disclosure. The present disclosure provides a GLP-2analog peptide composition of the present disclosure for use in a methodof increasing the urine sodium and/or potassium excretion in a subjectwith short bowel syndrome, wherein the GLP-2 analog peptide compositionof the present disclosure is for administration to the subject in atleast one therapeutically effective amount. The present disclosureprovides a GLP-2 analog peptide composition of the present disclosurefor use in the manufacture of a medicament for increasing the urinesodium and/or potassium excretion in a subject with short bowelsyndrome, wherein the GLP-2 analog peptide composition of the presentdisclosure is for administration to the subject in at least onetherapeutically effective amount.

In some aspects, the increase urine sodium and/or potassium excretioncan be at least about 5 mmol/day, or at least about 10 mmol/day, or atleast about 15 mmol/day, or at least about 20 mmol/day, or at leastabout 25 mmol/day, or at least about 30 mmol/day, or at least about 35mmol/day, or at least about 40 mmol/day, or at least about 45 mmol/day,or at least about 50 mmol/day.

The present disclosure provides a method for increasing intestinalabsorption of energy in a subject with short bowel syndrome, the methodcomprising administering to the subject at least one therapeuticallyeffective amount of a GLP-2 analog peptide composition of the presentdisclosure. The present disclosure provides a GLP-2 analog peptidecomposition of the present disclosure for use in a method of increasingthe intestinal absorption of energy in a subject with short bowelsyndrome, wherein the GLP-2 analog peptide composition of the presentdisclosure is for administration to the subject in at least onetherapeutically effective amount. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use inthe manufacture of a medicament for increasing the intestinal absorptionof energy in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount.

In some aspects, the increase intestinal absorption of energy can be atleast about 500 kJ/day, or at least about 600 kJ/day, or at least about700 kJ/day, or at least about 800 kJ/day, or at least about 900 kJ/day,or at least about 1000 kJ/day, or at least about 1100 kJ/day or at leastabout 1200 kJ/day.

The present disclosure provides a method for decreasing the energycontent of fecal output in a subject with short bowel syndrome, themethod comprising administering to the subject at least onetherapeutically effective amount of a GLP-2 analog peptide compositionof the present disclosure. The present disclosure provides a GLP-2analog peptide composition of the present disclosure for use in a methodof decreasing the energy content of fecal output in a subject with shortbowel syndrome, wherein the GLP-2 analog peptide composition of thepresent disclosure is for administration to the subject in at least onetherapeutically effective amount. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use inthe manufacture of a medicament for decreasing the energy content offecal output in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount.

In some aspects, the decrease in the energy content of the fecal outputcan be at least about 500 kJ/day, or at least about 600 kJ/day, or atleast about 700 kJ/day, or at least about 800 kJ/day, or at least about900 kJ/day, or at least about 1000 kJ/day, or at least about 1100 kJ/dayor at least about 1200 kJ/day.

The present disclosure provides a method for increasing carbohydrateand/or lipid absorption in a subject with short bowel syndrome, themethod comprising administering to the subject at least onetherapeutically effective amount of a GLP-2 analog peptide compositionof the present disclosure. The present disclosure provides a GLP-2analog peptide composition of the present disclosure for use in a methodof increasing carbohydrate and/or lipid absorption in a subject withshort bowel syndrome, wherein the GLP-2 analog peptide composition ofthe present disclosure is for administration to the subject in at leastone therapeutically effective amount. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use inthe manufacture of a medicament for increasing carbohydrate and/or lipidabsorption in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount.

In some aspects, the increase in carbohydrate and/or lipid absorptioncan be at least about 100 kJ/day, or at least about 200 kJ/day, or atleast about 300 kJ/day, or at least about 400 kJ/day, or at least about500 kJ/day, or at least about 600 kJ/day, or at least about 700 kJ/day,or at least about 800 kJ/day, or at least about 900 kJ/day, or at leastabout 1000 kJ/day, or at least about 1100 kJ/day or at least about 1200kJ/day.

The present disclosure provides a method for increasing proteinabsorption in a subject with short bowel syndrome, the method comprisingadministering to the subject at least one therapeutically effectiveamount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition ofthe present disclosure for use in a method of increasing proteinabsorption in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount. The present disclosure provides a GLP-2 analog peptidecomposition of the present disclosure for use in the manufacture of amedicament for increasing protein absorption in a subject with shortbowel syndrome, wherein the GLP-2 analog peptide composition of thepresent disclosure is for administration to the subject in at least onetherapeutically effective amount.

In some aspects, the increase in protein absorption can be at leastabout 100 kJ/day, or at least about 200 kJ/day, or at least about 300kJ/day, or at least about 400 kJ/day, or at least about 500 kJ/day, orat least about 600 kJ/day, or at least about 700 kJ/day, or at leastabout 800 kJ/day, or at least about 900 kJ/day, or at least about 1000kJ/day, or at least about 1100 kJ/day or at least about 1200 kJ/day.

The present disclosure provides a method for increasing body weight in asubject with short bowel syndrome, the method comprising administeringto the subject at least one therapeutically effective amount of a GLP-2analog peptide composition of the present disclosure. The presentdisclosure provides a GLP-2 analog peptide composition of the presentdisclosure for use in a method of increasing body weight in a subjectwith short bowel syndrome, wherein the GLP-2 analog peptide compositionof the present disclosure is for administration to the subject in atleast one therapeutically effective amount. The present disclosureprovides a GLP-2 analog peptide composition of the present disclosurefor use in the manufacture of a medicament for increasing body weight ina subject with short bowel syndrome, wherein the GLP-2 analog peptidecomposition of the present disclosure is for administration to thesubject in at least one therapeutically effective amount.

In some aspects, the increase in body weight can be at least about 0.5kg, or at least about 1.0 kg, or at least about 1.5 kg, or at leastabout 2.0 kg, or at least about 2.5 kg, or at least about 3.0 kg, or atleast about 3.5 kg, or at least about 4.0 kg, or at least about 4.5 kg,or at least about 5.0 kg.

The present disclosure provides a method for increasing lean body massin a subject with short bowel syndrome, the method comprisingadministering to the subject at least one therapeutically effectiveamount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition ofthe present disclosure for use in a method of increasing lean body massin a subject with short bowel syndrome, wherein the GLP-2 analog peptidecomposition of the present disclosure is for administration to thesubject in at least one therapeutically effective amount. The presentdisclosure provides a GLP-2 analog peptide composition of the presentdisclosure for use in the manufacture of a medicament for increasinglean body mass in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount.

In some aspects, the increase in lean body mass can be at least about0.5 kg, or at least about 1.0 kg, or at least about 1.5 kg, or at leastabout 2.0 kg, or at least about 2.5 kg, or at least about 3.0 kg, or atleast about 3.5 kg, or at least about 4.0 kg, or at least about 4.5 kg,or at least about 5.0 kg.

The present disclosure provides a method for decreasing fat mass in asubject with short bowel syndrome, the method comprising administeringto the subject at least one therapeutically effective amount of a GLP-2analog peptide composition of the present disclosure. The presentdisclosure provides a GLP-2 analog peptide composition of the presentdisclosure for use in a method of decreasing fat mass in a subject withshort bowel syndrome, wherein the GLP-2 analog peptide composition ofthe present disclosure is for administration to the subject in at leastone therapeutically effective amount. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use inthe manufacture of a medicament for decreasing fat mass in a subjectwith short bowel syndrome, wherein the GLP-2 analog peptide compositionof the present disclosure is for administration to the subject in atleast one therapeutically effective amount.

In some aspects, the decrease in fat mass can be at least about 0.5 kg,or at least about 1.0 kg, or at least about 1.5 kg, or at least about2.0 kg, or at least about 2.5 kg, or at least about 3.0 kg, or at leastabout 3.5 kg, or at least about 4.0 kg, or at least about 4.5 kg, or atleast about 5.0 kg.

The present disclosure provides a method for increasing theconcentration of L-citrulline in plasma in a subject with short bowelsyndrome, the method comprising administering to the subject at leastone therapeutically effective amount of a GLP-2 analog peptidecomposition of the present disclosure. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use in amethod of increasing the concentration of L-citrulline in plasma in asubject with short bowel syndrome, wherein the GLP-2 analog peptidecomposition of the present disclosure is for administration to thesubject in at least one therapeutically effective amount. The presentdisclosure provides a GLP-2 analog peptide composition of the presentdisclosure for use in the manufacture of a medicament for increasing theconcentration of L-citrulline in plasma in a subject with short bowelsyndrome, wherein the GLP-2 analog peptide composition of the presentdisclosure is for administration to the subject in at least onetherapeutically effective amount.

In some aspects, the increase in concentration in L-citrulline in plasmacan be at least about 5 μmol/L, or at least about 10 μmol/L, or at leastabout 15 μmol/L, or at least about 20 μmol/L.

The present disclosure provides a method for increasing urine volumeoutput in a subject with short bowel syndrome, the method comprisingadministering to the subject at least one therapeutically effectiveamount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition ofthe present disclosure for use in a method of increasing urine volumeoutput in a subject with short bowel syndrome, wherein the GLP-2 analogpeptide composition of the present disclosure is for administration tothe subject in at least one therapeutically effective amount. Thepresent disclosure provides a GLP-2 analog peptide composition of thepresent disclosure for use in the manufacture of a medicament forincreasing urine volume output in a subject with short bowel syndrome,wherein the GLP-2 analog peptide composition of the present disclosureis for administration to the subject in at least one therapeuticallyeffective amount.

In some aspects, the increase in urine volume output can be at leastabout 400 mL/day, or at least about 500 mL/day, or at least about 600mL/day, or at least about 700 mL/day, or at least about 800 mL/day, orat least about 900 mL/day, or at least about 1000 mL/day.

The present disclosure provides a method for decreasing oral fluidintake in a subject with short bowel syndrome, the method comprisingadministering to the subject at least one therapeutically effectiveamount of a GLP-2 analog peptide composition of the present disclosure.The present disclosure provides a GLP-2 analog peptide composition ofthe present disclosure for use in a method of decreasing oral fluidintake in a subject with short bowel syndrome, wherein the GLP-2 analogpeptide composition of the present disclosure is for administration tothe subject in at least one therapeutically effective amount. Thepresent disclosure provides a GLP-2 analog peptide composition of thepresent disclosure for use in the manufacture of a medicament fordecreasing oral fluid intake in a subject with short bowel syndrome,wherein the GLP-2 analog peptide composition of the present disclosureis for administration to the subject in at least one therapeuticallyeffective amount.

In some aspects, the decrease in oral fluid intake can be at least about400 mL/day, or at least about 500 mL/day, or at least about 600 mL/day,or at least about 700 mL/day, or at least about 800 mL/day, or at leastabout 900 mL/day, or at least about 1000 mL/day.

The present disclosure provides a method for decreasing parenteralsupport volume in a subject with short bowel syndrome, the methodcomprising administering to the subject at least one therapeuticallyeffective amount of a GLP-2 analog peptide composition of the presentdisclosure. The present disclosure provides a GLP-2 analog peptidecomposition of the present disclosure for use in a method of decreasingparenteral support volume in a subject with short bowel syndrome,wherein the GLP-2 analog peptide composition of the present disclosureis for administration to the subject in at least one therapeuticallyeffective amount. The present disclosure provides a GLP-2 analog peptidecomposition of the present disclosure for use in the manufacture of amedicament for decreasing parenteral support volume in a subject withshort bowel syndrome, wherein the GLP-2 analog peptide composition ofthe present disclosure is for administration to the subject in at leastone therapeutically effective amount.

In some aspects, the decrease in parenteral support volume can be atleast about 400 mL/day, or at least about 500 mL/day, or at least about600 mL/day, or at least about 700 mL/day, or at least about 800 mL/day,or at least about 900 mL/day, or at least about 1000 mL/day.

The present disclosure provides a method for decreasing plasmaaldosterone concentration in a subject with short bowel syndrome, themethod comprising administering to the subject at least onetherapeutically effective amount of a GLP-2 analog peptide compositionof the present disclosure. The present disclosure provides a GLP-2analog peptide composition of the present disclosure for use in a methodof decreasing plasma aldosterone concentration in a subject with shortbowel syndrome, wherein the GLP-2 analog peptide composition of thepresent disclosure is for administration to the subject in at least onetherapeutically effective amount. The present disclosure provides aGLP-2 analog peptide composition of the present disclosure for use inthe manufacture of a medicament for decreasing plasma aldosteroneconcentration in a subject with short bowel syndrome, wherein the GLP-2analog peptide composition of the present disclosure is foradministration to the subject in at least one therapeutically effectiveamount.

In some aspects, the decrease in plasma aldosterone concentration can beat least about 500 pmol/L, or at least about 750 pmol/L, or at leastabout 1000 pmol/L, or at least about 1250 pmol/L, or at least about 1500pmol/L, or at least about 1750 pmol/L, or at least about 2000 pmol/L, orat least about 2250 pmol/L, or at least about 2500 pmol/L, or at leastabout 2750 pmol/L, or at least about 3000 pmol/L.

In some aspects of the preceding methods, an increase can be an increaseof at least about 5%, or at least about 10%, or at least about 15%, orat least about 20%, or at least about 25%, or at least about 30%, or atleast about 35%, or at least about 40%, or at least about 45%, or atleast about 50%, or at least about 55%, or at least about 60%, or atleast about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% as compared to a control level.In some aspects, the control level is the amount prior to administrationof the GLP-2 analog peptide composition of the present disclosure.

In some aspects of the preceding methods, a decrease can be a decreaseof at least about 5%, or at least about 10%, or at least about 15%, orat least about 20%, or at least about 25%, or at least about 30%, or atleast about 35%, or at least about 40%, or at least about 45%, or atleast about 50%, or at least about 55%, or at least about 60%, or atleast about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 99% as compared to a control level.In some aspects, the control level is the amount prior to administrationof the GLP-2 analog peptide composition of the present disclosure.

In some aspects, the increase or decreases recited by the precedingmethods are after at least about four weeks of treatment with the GLP-2analog peptide composition of the present disclosure.

In some aspects, the short bowel syndrome can be short bowel syndromeintestinal insufficiency (SBS-II). In some aspects, the short bowelsyndrome can be short bowel syndrome intestinal failure.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Exemplary Embodiments

Embodiment 1. A composition comprising a sodium salt of apraglutide,wherein the sodium salt of apraglutide has a purity of no less than 95%,wherein apraglutide has the following structure:

Embodiment 2. The composition of embodiment 1, wherein the sodium saltof apraglutide has a purity of no less than 97%.

Embodiment 3. The composition of any one of the preceding embodiments,wherein the composition comprises no more than 3% of a Des-Gly⁴apraglutide impurity.

Embodiment 4. The composition of any one of the preceding embodiments,wherein the sum of Aspartimide³ apraglutide, Asp³³-OH apraglutide andDes-Ser⁷ apraglutide impurities in the composition is no more than 2%.

Embodiment 5. The composition of any one of the preceding embodiments,wherein the composition comprises no more than 2% of a [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] apraglutide impurity.

Embodiment 6. The composition of any one of the preceding embodiments,wherein the composition comprises no more than 1.5% of a β-Asp³apraglutide impurity.

Embodiment 7. The composition of any one of the preceding embodiments,wherein the composition comprises no more than 1% of a β-Asp³apraglutide impurity.

Embodiment 8. The composition of any one of the preceding embodiments,wherein the composition comprises no more than 1% of a D-His apraglutideimpurity.

Embodiment 9. The composition of any one of the preceding embodiments,wherein the composition comprises:

no more than 1% of a Asp³³-OH apraglutide impurity,

no more than 1% of a Des-Ser⁷ apraglutide impurity,

no more than 1% of a D-Aspartimide³ apraglutide impurity,

no more than 1% of a [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] apraglutideimpurity, and

wherein the sum of Des-Gly⁴ apraglutide and Aspartimide³ apraglutideimpurities in the composition is no more than 1%.

Embodiment 10. The composition of any of the preceding embodiments,wherein the sodium salt of apraglutide is provided as a lyophilizedpowder.

Embodiment 11. A pharmaceutical composition comprising the compositionof any one of the preceding embodiments.

Embodiment 12. The pharmaceutical composition of embodiment 11, furthercomprising at least one of glycine, L-histidine and mannitol.

Embodiment 13. A pharmaceutical composition of embodiment 10, whereinthe pharmaceutical composition comprises:

about 12.5 mg of apraglutide (sodium salt);

about 1.88 mg of glycine;

about 3.88 mg of L-histidine;

about 57.5 mg of mannitol.

Embodiment 14. The pharmaceutical composition of embodiment 12, whereinthe pharmaceutical composition is provided as a lyophilized powder.

Embodiment 15. A two-chamber powder syringe comprising the compositionof any one of embodiments 1-10 or the pharmaceutical composition of anyone of embodiments 11-14.

Embodiment 16. A method of making a GLP-2 analog peptide comprising:

a) performing solid phase peptide synthesis (SPPS) to synthesize theGLP-2 analog peptide on an Fmoc-Rink-amid-MethylBenzHydrilAmine(MBHA)-resin;

b) cleaving the synthesized GLP-2 analog peptide off the resin anddeprotecting the side chains of the synthesized GLP-2 analog peptide bytreating the resin with a solution comprising trifluoroacetic acid(TFA), water, and anisole;

c) purifying the synthesized GLP-2 analog peptide from step (b) byperforming a first preparative reversed-phase high performance liquidchromatography (RP-HPLC) purification using TFA-based mobile phases,thereby producing a solution comprising the GLP-2 analog peptide with apurity of no less than 90%;

d) purifying the product of step (c) by performing a second RP-HPLCpurification, using NaHCO₃-based mobile phases, thereby producingsolution comprising the GLP-2 analog peptide with a purity of no lessthan 97%.

Embodiment 17. The method of embodiment 16, further comprising:

e) further purifying the product from step (d) by performing a thirdRP-HPLC purification using NaOAc-based mobile phases, thereby producinga solution comprising the sodium salt of the GLP-2 analog peptide with apurity of no less than 97%.

Embodiment 18. The method of embodiment 17, further comprising:

f) adjusting the pH solution comprising the sodium salt of the GLP-2analog peptide to about pH 7.9 using 0.1% AcOH in water;

g) passing the product of step (f) through a filter with a pore size of0.2 μm;

h) lyophilizing the product of step (g), thereby producing lyophilizedsodium salt of the GLP-2 analog peptide with a purity of no less than97%.

Embodiment 19. The method of embodiment 16, further comprising:

c)(i) performing a decarboxylation of the synthesized GLP-2 analogpeptide by solubilizing the peptide in a solution comprising water andacetonitrile in ammonia buffer.

Embodiment 20. The method of embodiment 19, wherein the pH of thesolution comprising water and acetonitrile in ammonia buffer is adjustedto about pH 8.0.

Embodiment 21. The method of any one of embodiments 16-20, wherein step(a) comprises:

i) preparing a MBHA-resin on which the SPPS will be performed

ii) performing an initial Fmoc deprotection reaction followed by acoupling reaction to add a first Fmoc-protected amino acid to the resin,thereby forming a protected peptide on the resin;

iii) performing an Fmoc deprotection reaction followed by a couplingreaction to append at least one Fmoc-protected amino acid to theprotected peptide;

iv) repeating step iii until the GLP-2 analog peptide is synthesized onthe resin to produce a Fmoc-protected and side-chain protected GLP-2analog peptide linked to the resin;

v) performing an Fmoc deprotection reaction to produce a side-chainprotected GLP-2 analog peptide linked to the resin; and

vi) drying the side-chain protected GLP-2 analog peptide linked to theresin.

Embodiment 22. The method of embodiment 21, wherein step (a)(i)comprises:

(a1) washing the resin with a solution comprising dimethylformamide(DMF) and N,N-Diisopropylethylamine (DIEA) at 5 mL of solution per gramof resin under an N₂ atmosphere;

(b1) coupling a Rink amide linker to the resin in a solution comprising2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU), DIEA andHydroxybenzotriazole (HOBt) in DMF;

(c1) washing the product formed in step (b1) with DMF

(d1) performing a reduction reaction by contacting the resin with asolution comprising acetic anhydride (Ac₂O) and DIEA in DMF; and

(e1) washing the product formed in step (d1) with DMF.

Embodiment 23. The method of embodiment 21 or embodiment 22 whereinperforming an Fmoc deprotection reaction followed by a coupling reactioncomprises:

(a2) treating the resin with a solution comprising piperidine in DMF;

(b2) washing the resin with DMF;

(c2) washing the resin with a solution comprising DMF and oxyma;

(d2) contacting the resin with at least one Fmoc-protected amino acidand a first amount of a solution comprising diisopropylcarbodiimide(DIC) and ethyl cyanohydroxyiminoacetate (oxyma);

(e2) contacting the resin with a second amount of a solution comprisingDIC and oxyma; and

(f2) washing the product formed in step (e2) with DMF.

Embodiment 24. The method of embodiment 23, wherein the resin iscontacted with the second amount of a solution comprising DIC and oxymaabout 30 minutes after contacting the resin with the first amount of asolution comprising DIC and oxyma.

Embodiment 25. The method of embodiment 21 or embodiment 22 whereinperforming an Fmoc deprotection reaction followed by a coupling reactioncomprises:

(a2) treating the resin with a solution comprising piperidine and oxymain DMF;

(b2) washing the resin with DMF;

(c2) washing the resin with a solution comprising DMF and oxyma;

(d2) contacting the resin with at least one Fmoc-protected amino acidand a first amount of a solution comprising diisopropylcarbodiimide(DIC) and ethyl cyanohydroxyiminoacetate (oxyma);

(e2) contacting the resin with a second amount of a solution comprisingDIC and oxyma; and

(f2) washing the product formed in step (e2) with DMF.

Embodiment 26. The method of embodiment 25, wherein the resin iscontacted with a first amount of a solution comprising piperidine andoxyma in DMF for 15 minutes followed by contacting the resin with asecond amount of a solution comprising piperidine and oxyma in DMF for30 minutes.

Embodiment 27. The method of any one of embodiments 23 to 26, whereinthe at least one Fmoc-protected amino acid isFmoc-Gln(Trt)-Thr(ψ^(Me,Me)pro)-OH.

Embodiment 28. The method of any one of embodiments 23 to 26, whereinthe at least one Fmoc-protected amino acid is Fmoc-Gly(Tmb)-OH.

Embodiment 29. The method of any one of embodiments 23 to 26, whereinthe at least one protected amino acid is Boc-His(Trt)-Gly-OH.

Embodiment 30. The method of any one of embodiments 23-29, wherein themethod further comprises, between steps (e2) and (f2), performing acoupling test, wherein the coupling test is a Kaiser test.

Embodiment 31. The methods of any of embodiments 16-30, wherein theGLP-2 analog peptide is apraglutide.

Embodiment 32. A composition comprising the GLP-2 analog peptideproduced using the method of any of embodiments 16-31.

Embodiment 33. A method of treating short bowel syndrome associatedintestinal failure (SBS-IF) or short bowel syndrome associatedintestinal insufficiency (SBS-II) in a subject comprising administeringapraglutide, or pharmaceutically acceptable salt thereof, to thesubject,

wherein the apraglutide or pharmaceutically acceptable salt thereof isadministered at a dose of about 2.5 mg/week when the subject has a bodyweight of less than 50 kg, or

wherein the apraglutide or pharmaceutically acceptable salt thereof isadministered at a dose of about 5 mg/week when the subject has a bodyweight greater than or equal to 50 kg.

Embodiment 34. The method of embodiment 33, wherein the apraglutide, orpharmaceutically acceptable salt thereof, is administered bysubcutaneous injection.

Embodiment 35. The method of embodiment 33 or embodiment 34, wherein thesubject has colon-in-continuity, and wherein the apraglutide, orpharmaceutically acceptable salt thereof is administered for about 48weeks.

Embodiment 36. The method of embodiment 35, wherein the subject hasgreater than 50% colon-in-continuity.

Embodiment 37. The method of embodiment 33 or embodiment 34, wherein thesubject has at least one stoma, and wherein the apraglutide isadministered for about 24 weeks.

Embodiment 38. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof increasesthe intestinal absorption of dietary intake wet weight in a subjectrelative to an untreated or placebo treated subject.

Embodiment 39. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof decreasesfecal output in a subject relative to an untreated or placebo treatedsubject.

Embodiment 40. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof increasesabsolute urine volume output in a subject relative to an untreated orplacebo treated subject.

Embodiment 41. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof increasesintestinal absorption of sodium and potassium in a subject relative toan untreated or placebo treated subject.

Embodiment 42. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof increasessodium and potassium urine excretion in a subject relative to anuntreated or placebo treated subject.

Embodiment 43. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof increasesintestinal absorption of energy in a subject relative to an untreated orplacebo treated subject.

Embodiment 44. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof decreasesthe energy content of fecal output in a subject relative to an untreatedor placebo treated subject.

Embodiment 45. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof increasesintestinal absorption of carbohydrates, proteins, and lipids in asubject relative to an untreated or placebo treated subject.

Embodiment 46. The method of embodiment 33, wherein the administrationof apraglutide or a pharmaceutically acceptable salt thereof increasescitrulline concentration in a subject relative to an untreated orplacebo treated subject.

Embodiment 47. A sodium salt of apraglutide having a purity of no lessthan 95%, wherein apraglutide has the following structure:

Embodiment 48. The sodium salt of apraglutide of Embodiment 47, whereinthe sodium salt of apraglutide has a purity of no less than 97%.

Embodiment 49. The sodium salt of apraglutide of any one of thepreceding embodiments, wherein the sodium salt of apraglutide comprisesno more than 3% of a Des-Gly⁴ apraglutide impurity.

Embodiment 50. The sodium salt of apraglutide of any one of thepreceding embodiments, wherein the sum of Aspartimide³ apraglutide,Asp³³-OH apraglutide and Des-Ser⁷ apraglutide impurities in the sodiumsalt of apraglutide is no more than 2%.

Embodiment 51. The sodium salt of apraglutide of any one of thepreceding embodiments, wherein the composition sodium salt ofapraglutide no more than 2% of a [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)]apraglutide impurity.

Embodiment 52. The sodium salt of apraglutide of any one of thepreceding embodiments, wherein the sodium salt of apraglutide comprisesno more than 1.5% of a β-Asp³ apraglutide impurity.

Embodiment 53. The sodium salt of apraglutide of any one of thepreceding embodiments, wherein the sodium salt of apraglutide comprisesno more than 1% of a β-Asp³ apraglutide impurity.

Embodiment 54. The sodium salt of apraglutide of any one of thepreceding embodiments, wherein the sodium salt of apraglutide comprisesno more than 1% of a D-His apraglutide impurity.

Embodiment 55. The sodium salt of apraglutide of any one of thepreceding embodiments, wherein the sodium salt of apraglutide comprises:

no more than 1% of a Asp³³-OH apraglutide impurity,

no more than 1% of a Des-Ser⁷ apraglutide impurity,

no more than 1% of a D-Aspartimide³ apraglutide impurity,

no more than 1% of a [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] apraglutideimpurity, and

wherein the sum of Des-Gly⁴ apraglutide and Aspartimide³ apraglutideimpurities in the sodium salt of apraglutide is no more than 1%.

Embodiment 56. The sodium salt of apraglutide of any of the precedingembodiments, wherein the sodium salt of apraglutide is provided as alyophilized powder.

Embodiment 57. A pharmaceutical composition comprising the sodium saltof apraglutide of any one of the preceding embodiments.

Embodiment 58. The pharmaceutical composition of embodiment 57, furthercomprising at least one of glycine, L-histidine and mannitol.

Embodiment 59. A pharmaceutical composition of embodiment 58, whereinthe pharmaceutical composition comprises:

about 12.5 mg of apraglutide (sodium salt);

about 1.88 mg of glycine;

about 3.88 mg of L-histidine;

about 57.5 mg of mannitol.

Embodiment 60. The pharmaceutical composition of any one of embodiments57-59, wherein the pharmaceutical composition is provided as alyophilized powder.

Embodiment 61. A two-chamber powder syringe comprising the compositionof any one of embodiments 47-56 or the pharmaceutical composition of anyone of embodiments 57-60.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what is regarding as the invention.

Example 1: Apraglutide Manufacturing Process (I) Solid Phase PeptideSynthesis (Step 1)

The following is an exemplary method of the present disclosure for themanufacture of apraglutide at improved levels of purity relative topreviously described synthesis routes (e.g. U.S. Pat. No. 8,580,918).SPPS is the sequential synthesis of a peptide chain anchored on a solidsupport by repetition of a cycle encompassing the following steps:

1. Removal of the N-terminus Fmoc protecting group of the peptide resin

2. DMF washes

3. Couplings of Fmoc-AA-OH

4. Coupling test

5. DMF washes

This cycle is repeated until the peptide sequence is completed.

The α-amino groups of the amino acids are protected with thebase-sensitive 9-fluorenylmethyloxycarbonyl (Fmoc) group; the side chainfunctional groups are protected with acid-labile groups. All amino acidsderivatives used in the process are commercially available.

SPPS is the sequential synthesis of a peptide chain anchored on a solidsupport. In the synthesis, MBHA resin may be used to assemble thepeptide sequence. After swelling and washing the resin with DMF and thenwith DMF/DIEA under nitrogen atmosphere the Fmoc-Rink-amide linker maybe coupled using HBTU/DIPEA/HOBt in DMF. After coupling, the resin maybe washed with DMF and then acetylated using Ac₂O/DIPEA. A Kaiser testmay be carried out to check completion of the coupling.

After washing the resin with DMF, the Fmoc protected amino acids areeach coupled to the resin-bond peptide according to the following cycle:

1. The Fmoc-protecting group is removed with piperidine in DMF and theresin is washed thoroughly with DMF.2. The coupling is performed in DMF with variable amino acid equivalentsusing DIC/oxyma for activation.3. Coupling of amino acids is monitored by using the Ninhydrin assay,which is performed during each synthesis cycle.

At the end of the assembly, after the last amino acid has been coupledand deprotected, the resin is washed with DMF and isopropanol, and driedunder vacuum.

Cleavage of the Peptide from the Resin and Deprotection (Step 2)

The protected peptide may be simultaneously cleaved from the resin anddeprotected by treatment with a mixture TFA/water/anisole. MTBE issubsequently added to the peptide/TFA slurry to precipitate the crudepeptide in the presence of cleaved resin. The obtained crude peptide isfiltered, washed with MTBE and dried under vacuum to constant weight.

Decarboxylation Reaction (Step 3a)

The crude peptide is solubilized in a mixture of H₂O/ACN (80:20 ratio)in ammonia buffer. The solution is adjusted to target pH≥7 using 25%NH₄OH in H₂O. The decarboxylation reaction is maintained at RoomTemperature® for 24 hours. The crude peptide is washed with a solutionof H₂O/ACN (80:20 ratio) in ammonia buffer at about pH 10 and stored atRoom Temperature®.

Purification by Preparative RP-HPLC (Step 3b)

The crude peptide is dissolved in a mixture of water/acetonitrile/NH₄OH.This solution is diluted with acetic acid and then filtered.

The primary purification is conducted on preparative RP-HPLC withNaHCO₃/H₂O/CH₃CN as eluent. The elution from the column is monitored byUV and the fractions obtained are analyzed by RP-HPLC. Fractions meetingthe monitoring criteria are mixed in the combined pool. Fractions notmeeting the monitoring criteria may be recycled by repeating thepurification step. The purity of the pool is controlled by analyticalRP-HPLC.

Sodium Salt Conversion by Preparative RP-HPLC (Step 4)

This step may be conducted to exchange the counter ion of the peptidefrom a TFA anion to a sodium cation through a pH change and to furtherpurify the peptide. The combined pool from Step 3 is diluted in waterand re-purified by preparative RP-HPLC using NaOAc eluent.

The purified peptide solution is subsequently subject to evaporationunder vacuum to reduce acetonitrile in the solution. The purifiedpeptide solution is then adjusted to target pH 7.9 using 0.1% AcOH.

The pure pool may be concentrated and freeze-dried. The purity of thepool is analyzed by RP-HPLC.

Freeze-Drying and Packaging (Step 5)

Prior to lyophilization, the purified peptide in solution is filteredthrough a 0.2 μm membrane. The lyophilization is carried out at lowpressure. The resulting lyophilized final peptide is packed under argon.The lyophilized apraglutide is controlled according to the apraglutidespecification.

Reprocessing

Lyophilized apraglutide that does not fulfil the criteria established inthe apraglutide specification may be subjected to re-purification.

Re-purification may be carried out after reconstitution of the peptideby repeating the purification step(s) and counterion conversion step, asdescribed above.

After re-purification, the material is lyophilized according to theprocedure described above.

Lyophilized apraglutide that does not fulfil the criteria established inthe apraglutide specification may be subjected to re-lyophilization.

Re-lyophilization may be carried out after reconstitution of the peptideby repeating the lyophilization step, as described above.

Impurities

In some aspects of the methods of the present disclosure, theapraglutide is substantially free from organic impurities including, butnot limited to, residues of the reagents and materials (includingby-products) used in the manufacturing process.

In some aspects of the methods of the present disclosure, theapraglutide is substantially free from non-peptide impurities.

In some aspects of the methods of the present disclosure, theapraglutide is substantially free from residual solvents.

The peptide impurities, including but not limited to, Des-Gly⁴apraglutide, Aspartimide³ apraglutide, Asp³³-OH apraglutide, Des-Ser⁷apraglutide, and [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)] apraglutide may beobserved in the apraglutide composition.

Apraglutide for injection may be a lyophilized powder stored at the longterm storage condition (5°±3° C.). The stability results provided herein(Table 5) demonstrate that no degradation has been observed at thelong-term storage condition.

A test of bacterial endotoxins may be preferred to ensure themicrobiological quality after manufacture, packaging, storage anddistribution of the apraglutide composition, as required for aparenteral apraglutide composition. The test is performed according toPh. Eur. 2.6.14/USP <85>.

Chemical Characteristics of Product

The compound of the present disclosure is a 33 amino acid syntheticpeptide analogue of glucagon-like peptide-2 (GLP-2) that acts as aselective, full agonist of the GLP-2 receptor with potency andselectivity comparable to native GLP-2.

The peptide of the disclosure a linear peptide. The sequence of thepeptide contains one D stereoisomer amino acid (D-phenylalanine), oneunnatural amino acid (Norleucine) and two achiral amino acids (glycine);all other amino acids are L-configuration. The peptide of the disclosureis synthesized as a single enantiomer with all stereo-centers of definedchirality. It may be isolated in its sodium salt form with some residualwater as a natural constituent.

The appearance of the disclosed peptide is a white to off-whitehomogenous powder. It may be isolated by reversed phase chromatographypurification and subsequently lyophilized; no crystalline or polymorphicforms are known.

The solubility of the disclosed peptide in purified water is above 100mg/mL.

The pH of a solution of the disclosed peptide in water at 100 mg/mL is7.6 to 8.4. The calculated isoelectric point is 4.3a.

The optimum solubility and chemical stability of apraglutide wasobserved between pH 8.0 to 8.5.

In-Process Controls

In some embodiments, the purity of purified peptide pool as measured byRP-HPLC is greater than 95%, 97% or 99%. In some embodiments, the purityof purified peptide pool as measured by RP-HPLC is greater than 95%.

In some aspects, the in-process controls presented in Table 4 can be usein the methods of the present disclosure. In some aspects, thein-process controls presented in Table 4 can have a purity that ispresented in the “Acceptance Criteria” column of Table 4:

TABLE 4 In-Process Control Analytical Test Steps in the ProcessPrinciple Acceptance Criteria Purity of the crude Step 2: Cleavage ofthe RP-HPLC Overall purity ≥ 50.0% (area) peptide peptide from the resinand side chain deprotection Purity of purified Step 3: Purification byRP-HPLC Purity ≥ 95% peptide pool by RP- preparative RP-HPLC Sum ofDes-Gly⁴ apraglutide and HPLC Aspartimide³ apraglutide ≤ 1.0% Asp33-OHapraglutide ≤ 1.0% Des-5er7 apraglutide ≤ 1.0% [Trp25,2-(2,4,6-trimethoxyphenyl)] apraglutide ≤ 2.0% D-Aspartimide apraglutide≤ 2.0% Purity of peptide Step 4: Sodium salt RP-HPLC Purity ≥ 95% sodiumsalt conversion by preparative Sum of Des-Gly⁴ apraglutide and RP-HPLCAspartimide³ apraglutide ≤ 1.0% Asp33-OH apraglutide ≤ 1.0% Des-5er7apraglutide ≤ 1.0% [Trp25, 2-(2,4,6-trimethoxyphenyl)] apraglutide ≤2.0% D-Aspartimide³ apraglutide ≤ 2.0%

The manufacture process described in this example is herein referred toas Process A. Table 2a shows the purity of Apraglutide product producedusing Process A, as well as the level of major contaminants. As shown inTable 2a and Table 2b, Process A can yield Apraglutide that has a purityof no less than 95%, and low levels of contaminants. Moreover, Process Aexhibits product yields ranging from 15% to 22%.

Example 2: In Vitro Measurement of GLP-2 Agonist Activity

To determine activities of GLP-2 agonists of the present disclosure onthe hGLP-2 receptor, a transcriptional reporter gene assay is used. Twoconstructs are transiently transfected into a human embryonic kidneycell line (HEK-293): an hGLP-2 receptor expression DNA construct and areporter DNA construct containing intracellular cAMP-responsive promoterelements regulating expression of firefly luciferase. (See for exampleHimmLer et al., J. Recept. Res., (1993), 13, 79-74 for further guidanceon this assay.)

Cells are exposed for 5 hours to serial dilutions of compounds dilutedhalf-log per dose. After exposure to compounds, cells are lysed,luciferase activity is determined, and compound efficacies and EC50values are determined by non-linear regression analysis. hGLP-2, anaturally occurring 33-amino acid peptide ligand, is used as an internalcontrol in each experiment.

Example 3: Formulation

To generate a formulation suitable for subcutaneous injection,apraglutide is aseptically manufactured and freeze-dried into a powdertogether with the pharmaceutical excipients glycine, L-histidine, andmannitol. The freeze-dried powder is reconstituted with sterile water orbuffer for injection.

To develop a lyophilized formulation, a screening study was conductedevaluate the optimal bulking agent and excipients to achieve the desiredphysical stability of apraglutide in the liquid phase. Initial testingof the bulking agents sucrose and mannitol demonstrated that both weresuitable for the lyophilization process and in terms of reconstitutiontime, water content and visual inspection of the lyophilized products.The effect of concentrations and combination of the stabilizers with thetwo bulking agents, sucrose and mannitol, was investigated. Glycine andTris were tested as buffering agents at concentrations of 20 Mm (1.50mg/mL for Glycine and 2.4 mg/mL for Tris) and 40 mM (3.0 mg/mL forGlycine and 4.8 mg/mL for Tris), pH 8.2 to 8.5. All formulations weretested with or without the presence of L-histidine at a 20 mM (3.10mg/mL) concentration and with either mannitol or sucrose as bulkingagent. A total of eleven formulations were prepared using factorialdesign. The formulations were physically challenged by placement on anorbiting table and at an accelerated temperature of 40° C. for 3 days.Physical stability of the formulations was assessed by size exclusionchromatography, optical density and viscosity; chemical stability ofapraglutide was assessed by reversed phase liquid chromatography andsize exclusion chromatography. The chemical and physical stability offormulations containing glycine in combination with histidine weresuperior to formulations containing Tris-buffer. Mannitol and sucrosewere both suitable as bulking agents; however, mannitol was chosen dueto the better processability during lyophilization.

As the effect of glycine concentration could not be concluded from thescreening study, a laboratory scale stability study was set up with twoformulations containing glycine at concentrations of 20 Mm (1.50 mg/mL)and 40 Mm (3.0 mg/mL), respectively. Both formulations contained1-histidine, mannitol, apraglutide, and pH adjusted to 8.3. Theformulations were freeze-dried and followed for 3 months at −20° C., 5°C., 25° C. and 40° C. The conclusions were that at −20° C., 5° C., and25° C., both formulations were chemically and physically stablethroughout the testing period of 3 months. At 40° C., both formulationsshowed signs of decreased physical stability at 3 months. Theformulation containing the lower concentration of 20 mM glycine had apreferred higher degree of crystallinity of the mannitol when assessedby X-ray powder diffraction. Hence, 20 mM glycine was chosen for theformulation.

Example 4: Citrulline as a Marker for Apraglutide Effect

Plasma citrulline was used as a pharmacodynamic (“PD”) marker todescribe the effect of apraglutide. Similar to the pharmacokinetic(“PK”) model development, the PK/PD model was developed for the healthyindividuals from studies GLY-101-2015 and TA799-002 only. Fordevelopment of the PD model, the PD observations were fitted togetherwith the PK observations using the final PK model with its structure,residual error-, covariates- and random effects-models. The startingmodel was a turnover model where plasma citrulline was constantlysynthesized and degraded with the synthesis rate ksyn and thedegradation rate kdeg. Plasma apraglutide stimulated citrullinesynthesis via a sigmoid Emax relationship with the baseline citrullinelevel RO, the half-maximal effect at the concentration EC50, the maximaleffect Emax, and a Hill coefficient gamma. It was assumed thatcitrulline turnover was in steady state such that citrulline synthesiswas determined solely by citrulline baseline and degradation. Thepopulation PK/PD of plasma apraglutide was established with data fromtwo studies in healthy volunteers and two studies with SBS patients thattested doses between 2.5 and 50 mg SC. The concentration of apraglutidefollowed two-compartmental and dose-nonlinear kinetics. Apraglutideclearance was estimated to 16.8 L/day, the central volume ofdistribution to 31.5 L for a 70-kg individual receiving a 5 mg SC dose.The half-life was estimated to 1.3 days. The volume of distribution andthe clearance were body weight dependent with coefficientsβ_(V1/F,BW)=1.94, and β_(Cl/F,BW)=1.8. These coefficients indicated astrong body weight dependence. Zero-order absorption duration wasdependent on dose with a coefficient of β_(Tk0,Dose)=0.249.

The effect of plasma apraglutide on plasma citrulline was found to besigmoidal with a citrulline saturation appearing at doses of 5 mg. Thisphenomenon was described with a citrulline turnover model whosesynthesis was stimulated according to an E_(max) model by apraglutide.Citrulline baseline was found to be 5.12 μg/mL, while the maximal effectwas 0.626, and the half-maximal effective apraglutide concentration was13.7 ng/mL.

The population PK/PD model was used to predict plasma apraglutideexposure and plasma citrulline trough concentration-time profiles withand without inter-individual variability for individuals with bodyweights between 40 and 120 kg receiving weekly doses of 2.5, 5, or 10 mgSC. Predictions without inter-individual variability showed that theeffect of apraglutide on citrulline begins to saturate for a 40 kgindividual receiving weekly 5 mg apraglutide SC, i.e. with decreasingbody weight the same apraglutide dose resulted in a decreasing effect oncitrulline. This saturation effect was found to be even more pronouncedfor individuals receiving weekly 10 mg apraglutide SC. Predictions withinter-individual variability displayed a large variability in plasmacitrulline concentration-time profiles and plasma citrulline troughlevels. Apraglutide exposure for body weights between 40 and 120 kg andapraglutide SC doses of 2.5, 5, or 10 mg was compared to post-hocexposure estimates of SBS patients. Bracketing at a body weight of 50 kgbetween doses of 2.5 and 5 mg was simulated. This bracketing variantresulted in apraglutide exposures within the range of post-hoc AUCestimates of SBS patients receiving weekly 5 mg apraglutide SC. Asclinical biomarker, urinary output was correlated to post-hoc estimatesof plasma apraglutide exposure and plasma citrulline trough levels inSBS patients. It was found that apraglutide exposure and citrullinetrough levels only correlated weakly with urinary output.

Plasma citrulline, a biomarker for enterocytic mass, is increased afterGLP-2 analog administration in human clinical trials.

The pharmacokinetic/pharmacodynamic (PK/PD) relationship betweenapraglutide, a novel long-acting GLP-2 analog in development for shortbowel syndrome, and citrulline was evaluated in a randomized,double-blind, parallel arm, placebo-controlled, multiple dose study. 23healthy adult volunteers received 6 weekly subcutaneous doses ofapraglutide (1, 5, or 10 mg) or placebo and were followed for a further6 weeks after the last dose. Blood collections were controlled for diet,lifestyle, and diurnal effects. L-citrulline was quantified using avalidated LC-MS method. PK data underwent non-compartmental analysis andPD data were analyzed by ANCOVA.

PK parameters indicated a half-life of 72 hours. Increases in citrullinewere observed 2 days after the first apraglutide dose and maximal effectwas achieved in most subjects after 4 or 7 days. Mean citrulline levelswere elevated from baseline in all apraglutide arms and remainedelevated during the 6-week treatment period. Citrulline increases weresignificantly greater with apraglutide 5 mg and 10 mg than withapraglutide 1 mg versus placebo (Table 5). There were no statisticallysignificant differences between 5 and 10 mg apraglutide dose levels.Plasma citrulline levels remained elevated for 10 to 17 days after thefinal apraglutide dose. Apraglutide was safe and well-tolerated with noserious AEs.

TABLE 5 Difference in citrulline Apraglutide dose (mg) (μg/mL) vsplacebo P-value 1 0.31 [95% CI: −0.4371; 0.3910 1.0663] 5 1.26 [95% CI:0.5037; 0.0025 2.0112] 10 1.63 [95% CI: 0.8809; 0.0002 2.3878]

Apraglutide was safe and well tolerated, and displayed a PD responselonger than plasma exposure. These PK and PD data confirm the potentialfor once-weekly subcutaneous dosing of apraglutide.

Example 5: Phase II Human Clinical Trial Results of Administration ofApraglutide Compositions of the Present Disclosure

The following non-limiting example describes results from a Phase 2clinical trial in which subjects with SBS-IF were treated with theapraglutide formulations of the present disclosure. This Phase 2 trialinvestigated the safety and efficacy of 5 and 10 mg apraglutide inpatients with SBS-IF.

Without wishing to be bound by theory, the beneficial effects of GLP-2analogues in SBS patients may include dynamic changes in body fluidhomeostasis, which may be considered when evaluating treatment outcomes.Improved intestinal absorption results in decreased fecal output. Sincelarge amounts of the intestinally absorbed water and sodium is excretedby the kidneys though the systemic circulation, an increased absorptionin SBS patients can lead to an increase in urine volume production andsodium excretion. Therefore, in general, the hydration state of the SBSpatient can be monitored by measuring urine volume and urine sodium.

Eight adult patients with SBS-IF were treated with apraglutidecompositions of the present disclosure according to Table 6. Briefly,patients with SBS-IF were administered 5 mg apraglutide of the presentdisclosure or placebo once a week for four weeks. The patients were thenadministered 10 mg apraglutide of the present disclosure once a week forfour weeks. Additionally, there was a washout period of 6-10 weeksbetween treatments.

TABLE 6 Part A: Washout 1 Part A: Washout 2 Part B: Treatment (6-10Treatment (6-10 Treatment period 1 weeks period 2 weeks period 3 (4weeks) after last (4 weeks) after last (4 weeks) Randomized dose)Randomized dose) Open-label Placebo Placebo 10 mg or 5 mg or 5 mgapraglutide apraglutide apraglutide subcutaneously subcutaneouslysubcutaneously once weekly once weekly once weekly

Results

As part of the trial, a total of 12 patients were screened, and out ofthem, 8 were randomized for treatment. All 8 patients continued in theadditional treatment period in an open-label regimen with 10 mgapraglutide (Part B). One patient discontinued the trial after firstdrug administration in the Part B treatment period due to exhaustionfrom trial procedures and a perceived lack of effect. The patientprovided data for the immediate measurements but not post-treatment.Eight patients comprised the safety analysis set and the full analysisset. Demographics and baseline characteristics of the patients aresummarized in Table 7. Data in Table 7 are mean (SD) or N (%) andparenteral support (PS) was scheduled PS at trial entry based on aweekly average.

Three patients had previously been treated with a GLP-2 analogue in aclinical trial (≥6 months ago). Individual patient plots illustratedthat all effects reverted to baseline after the washout period; thus, nocarryover effect was observed.

TABLE 7 Age (years) 59.1 (13.8) Sex Female 4 (50%) Male 4 (50%) Weightat baseline 75.6 (15.5) Body-mass index (kg/m2) 24.8 (3.9) Race, white 8(100%) Parenteral support volume (mL/day) 3,309 (1,903) Parenteralsupport energy (kJ/day) 4,665 (3,852) Urine volume output (mL/day) 2,031(1,112) Oral fluid intake (mL/day) 2,799 (1,289) Urine sodium excretion73 (94) Plasma citrulline levels (μmol/L) 33 (30) Cause of resectionCrohn's disease 2 (25%) Mesenteric vascular disease 3 (37.5%) Surgicalcomplications to ulcerative colitis 1 (12.5%) Surgical complications 2(25%) Disease characteristics Small bowel length (cm) 109 (127)End-jejunostomy 6 (75%) lleostomy 2 (25%) Colon in continuity 0Concomitant medication Proton-pump inhibitor 7 (87.5%) Opioids or opioidagonists 4 (50%) Loperamide 3 (37.5%) Total (N = 8)

Safety Results

Common related adverse events are reported in Table 8 (data are N or N(%)). Adverse events that occurred in >2 patients and included polyuria(n=7), stoma complications (n=6), gastrointestinal stoma complication(n=5), gastrointestinal stoma output decreased (n=6), gastrointestinalstoma output abnormal (n=5), decreased thirst (n=5), edema (n=4),increased weight (n=3) and decreased appetite (n=3). Three patientsexperienced injection site reactions. All injection site reactionsoccurred with active treatment; one patient had an injection sitereaction in the 5 mg dose group, and three patients had an injectionsite reaction in the 10 mg dose group. A total of 8 cases of seriousadverse events were reported by 5 patients. None of these wereconsidered related to the trial drug. Two serious adverse eventsinvolved mechanical complications related to the tunneled central venouscatheter used by the patients for PS administration, and both requiredhospitalization for catheter replacement. Six serious adverse eventswere catheter related blood stream infections (CRBSIs). One patient hadfour recurrent events of CRBSI. Serious adverse events were distributedequally between placebo and treatment periods. Most patients had atleast one treatment related adverse event, all of which were mild tomoderate with no obvious difference between the active dose levels.

TABLE 8 Placebo 5 mg 10 mg Total (N = 8) (N = 8) (N = 8) (N = 8) Anyrelated adverse events 8 8 8 8 (100%) Polyuria 1 4 6 7 (88%)Gastrointestinal stoma 0 3 6 6 (75%) output decreased Stoma complication0 6 6 6 (75%) Gastrointestinal stoma 0 5 5 5 (63%) complicationGastrointestinal stoma 0 4 4 5 (63%) output abnormal Thirst decreased 03 4 5 (63%) Edema 0 2 2 4 (50%) Increased weight 0 1 2 3 (38%) Decreasedappetite 0 1 2 3 (38%)

There were no trial discontinuations or reductions due to relatedadverse events. One patient omitted the third dose of apraglutide duringthe placebo period due to peripheral edema. No safety concerns wereraised from vital signs, blood samples, ECG, dipstick urinalysis or bodyweight. There were no deaths.

Three patients developed anti-apraglutide antibodies during the trial.Two patients tested positive for anti-apraglutide antibodies at the endof the 5 mg treatment period, and one at the end of the 10 mg treatmentperiod. One patient who had positive antibodies at the end of the 5 mgtreatment period, also had positive antibodies in the end of the 10 mgtreatment period. For the two patients who developed positive antibodiesduring the 5 mg treatment period, they were negative after a 6-10 weekwashout period. For the two patients with positive antibodies at the endof the 10 mg treatment, both were still positive after a 4-6 weekswashout period, but the titres were lower than at the end of the 10 mgtreatment period. No effects of anti-apraglutide antibodies weredetected on the PK profile, the pharmacodynamic response to apraglutide,or on the number or duration of adverse events.

Urine Volume Output and Urine Sodium Excretion: Changes from Baseline toEnd of Treatment (Day 27-29)

The individual changes from baseline to end of each treatment inabsolute urine volume output are plotted in FIG. 5, including the meanchange for each treatment. The mean change differed from the adjustedmean calculated in the statistical analyses, which compares treatmentsto placebo. For the statistical analysis of Part A+B, 5 mg apraglutidepromoted the increase of absolute urine volume output by an adjustedmean of 711 mL/day (95% CI 132 to 1,289; P=0.021) compared to placebo,corresponding to a daily increase of 48% (95% CI 12 to 84; P=0.014), asshown in Table 9. For the statistical analysis of Part A, 5 mgapraglutide promoted the increase of absolute urine volume output by anadjusted mean of 714 mL/day (95% CI 490 to 939; P=0.002), correspondingto a daily increase of 49% (95% CI 4 to 94; P=0.041), as shown in Table10. Treatment with 10 mg apraglutide promoted the increase of absoluteurine volume output by an adjusted mean of 795 mL (95% CI 195 to 1,394;P=0.014) compared to placebo. The corresponding change in relative urineproduction was 34% (95% CI −4 to 71; P=0.072). No difference was seenbetween the 5 mg and 10 mg dose groups, as shown in Table 9. In Table 9,the data are shown as an adjusted mean (95% CI), calculations are basedon changes from baseline to end of treatment or near end of treatment ofindividual dose groups, and N denotes the number of patients in the fullanalysis set. The results from the statistical analysis of Part A+B arepresented graphically in FIG. 6.

TABLE 9 Analysis Part A + B 5 mg vs placebo 10 mg vs placebo 5 mg vs 10mg (N = 8) (N = 8) (N = 8) Absolute urine 711 (132 to 1,289); 795 (195to 1,394); 84 (−514 to 682); output (mL/day) P = .021 P = .014 P = .761Relative urine 48(12 to 84); 34 (−4 to 71); −14 (−51 to 23); output (%)P = .014 P = .072 P = .420 Urine sodium 56 (−10 to 123); 88 (20 to 156);32 (−37 to 101); excretion P = .087 P = .017 P = .325 (mmol/day) Urinesodium 166 (−342 to 675); 432 ( −87 to 951); 266 (−266 to 798);excretion (%) P = .478 P = .092 P = .287 PS volume −89 (−543 to 366);−469 (−941 to 4); −380 (−851 to 91); (mL/day) P = .676 P = .052 P = .103Relative PS −13 (−36 to 9); −28 (−51 to −4); −15 (−38 to 9); volume (%)P = .225 P = .025 P = .195 Oral fluid −244 (−512 to 23); −363 (−641 to−86); −119 (−396 to 157); intake (mL/day) P = .070 P = .015 P = .362Relative oral −9 (−18 to 1); −15 (−25 to −5); −7 (−17 to 3); fluidintake (%) P = .072 P = .006 P = .169

The individual changes from baseline to end of each treatment inabsolute urine sodium excretion are plotted in FIG. 7. For thestatistical analysis of Part A+B, 5 mg apraglutide increased urinesodium excretion compared to placebo by an adjusted mean of 56 mmol/day(95% CI −10 to 123; P=0.087), as shown in Table 9. For the statisticalanalysis of Part A, 5 mg apraglutide increased urine sodium excretioncompared to placebo by an adjusted mean of 66 mmol/day (95% CI −69 to201; P=0.171), as shown in Table 10. In Table 10, the data are shown asan adjusted mean (95% CI), calculations are based on changes frombaseline to end of treatment of individual dose groups, and N denotesthe number of patients in the full analysis set. In the 10 mg dosegroup, absolute urine sodium excretion was increased by an adjusted meanof 88 mmol/day (95% CI 20 to 156; P=0.017) compared to placebo, as shownin Table 9. As shown in Tables 9 and 10, relative urine sodium excretionwas not changed following apraglutide treatment. As further shown inTable 9, no difference was found between the 5 mg and 10 mg dose groups.

TABLE 10 Analysis Part A 5 mg vs placebo (N = 8) Absolute urine output(mL/day) 714 (490 to 939); P = .002 Relative urine output (%)   49 (4 to94); P = .041 Urine sodium excretion (mmol/day)  66 (−69 to 201); P =.171 Urine sodium excretion (%) 189 (−350 to 729); P = .270  PS volume(mL/day) −94 (−344 to 156); P = .356  Relative PS volume (%)  −13 (−41to 15); P = .276 Oral fluid intake (mL/day) −242 (−560 to 76); P = .103 Relative Oral fluid intake (%)   −9 (−18 to 1); P = .068Urine Volume Output and Urine Sodium Excretion: Changes from Baseline toImmediately after First Treatment Injection (Day 1-3)

The individual changes from baseline to immediately after firsttreatment injection in absolute urine output and urine sodium excretionare shown plotted in FIGS. 8 and 9, respectively. No immediate changeswere observed for the 5 mg or 10 mg dose group compared to placebo, andthere were no differences between the active dose groups, as shown inTable 11. In Table 11, the data are shown as an adjusted mean (95% CI)and calculations are based on changes from baseline to immediately afterfirst treatment injection of individual dose groups, and N denotes thenumber of patients in the full analysis set.

TABLE 11 Analysis Analysis Part A + B Part A 5 mg vs placebo 10 mg vsplacebo 5 mg vs 10 mg 5 mg vs placebo (N = 8) (N = 8) (N = 8) (N = 8)Absolute urine −38 (−456 to 381); −64 (−500 to 372); −26 (−462 to 409);−48 (−696 to 601); output (mL/day) P = .845 P = .751 P = .896 P = .831Relative urine 6 (−24 to 35); −6 (−37 to 24); −12 (−43 to 19); 5 (−38 to48); output (%) P = .678 P = .655 P = .402 P = .729 Urine sodium 3 (−17to 23); 3 (−17 to 22); −1 (−22 to 20); 3 (−146 to 152); excretion P =.705 P = .768 P = .930 P = .823 (mmol/day) Urine sodium 65 ( −36 to166); 22 ( −75 to 119); −43(−149 to 63); 64 (−306 to 433); excretion (%)P = .176 P = .617 P = .375 P = .272Oral Fluid Intake: Changes from Baseline to Near End of Treatment (Day20-22)

At days 20-22, treatment with 10 mg apraglutide decreased absolute andrelative oral fluid intake by an adjusted mean of 363 mL/day (95% CI−641 to −86; P=0.015) and −15% (95% CI −25 to −5; P=0.006) respectively.For the analysis of Part A+B, a similar trend was seen for the 5 mg dosegroup with an adjusted mean reduction of 244 mL/day (95% CI −512 to 23;P=0.070), as shown in Table 9, and for the analysis of Part A with amean reduction of 242 mL/day (95% CI −560 to 76; P=0.103), as shown inTable 10. Further, as shown in Table 9, no difference was found betweenthe active dose groups.

PS Volume: Changes from Baseline to Near End of Treatment (Day 20-22)

At days 20-22, 10 mg apraglutide decreased relative daily PS volume by−28% (−51 to −4; P=0.025) compared to placebo. A similar trend was seenfor the absolute change in daily PS volume which decreased by 469 mL/day(−941 to 4; P=0.052), as shown in Table 9. No changes in PS volume wereobserved for the 5 mg dose group, as shown in Tables 9 and 10, and nodifference was seen between the 5 mg and 10 mg dose groups, as shown inTable 9.

Fluid Composite Effect: Changes from Baseline to Near End of Treatment(Day 20-22)

A post-hoc analysis for Part A+B was performed to calculate the fluidcomposite effect, defined as the sum of increase urine production (day27-29), reduction in PS volume and reduction in spontaneous oral fluidintake (day 20-22). Spontaneous oral fluid intake and PS volumereduction were assessed near the end of treatment period, since theywere kept unchanged during urine collections. Administration of 5 mg and10 mg apraglutide increased the fluid composite effect compared toplacebo by 1,036 mL/day (95% CI 262 to 1,810; P=0.014) and 1,630 mL/day(95% CI 827 to 2,433; P=0.001), respectively. There was no differencebetween the two doses; the estimated difference was 594 (95% CI −207 to1,396; P=0.129). Individual plots of the fluid composite effect togetherwith urine volume output at end of treatment are shown in FIGS. 10A-C.

Hydration Status Parameters

In a post-hoc analysis of Part A+B apraglutide, 5 mg and 10 mg reducedplasma aldosterone by 2,894 pmol/L (95% CI −6247 to 458; P=0.083) and3,045 pmol/L (95% CI −6,460 to 370; P=0.075), respectively. Apraglutidedid not change other hydration parameters including creatinine, urea,hematocrit, albumin, protein and CO₂ total, as shown in Table 12. InTable 12, the data are shown as an adjusted mean (95% CI), calculationsare based on changes from baseline to end of treatment of individualdose groups, and N denotes the number of patients in the full analysisset.

TABLE 12 Analysis Part A + B 5 mg vs placebo 10 mg vs placebo 5 mg vs 10mg (N = 8) (N = 8) (N = 8) Aldosterone −2,894 (−6,247 to 458); −3,045(−6,460 to 370), −151 (−3,570 to 3,268); (pmol/L) P = .083 P = .075 P =.924 Creatinine −9.7(−25.8 to 6.4); −5.1 (−22.1 to 11.9); 4.6 (−12.3 to21.5); (umol/L) P = .214 P = .525 P = .567 Urea −0.9 (−2.4 to 0.6); −0.6(−2.2 to 1.0); 0.3 (−1.3 to 1.9); (mmol/L) P = .217 P = .425 P = .691Hematocrit 0.6 (−0.2 to1.4); 0.2 (−0.7 to 1.0); −0.4 (−1.2 to 0.4);(vol/fr) P = .151 P = .675 P = .326 Albumin −0.0 (−1.8 to 1.8); 1.0(−0.9 to 2.9); 1.0 (−0.8 to 2.9); (g/L) P = .996 P = .260 P = .248Protein −0.4 (−4.2 to 3.4); −0.7 (−4.7 to 3.3); −0.3 (−4.5 to 3.9);(g/L) P = .824 P = .704 P = .874 CO2 total 0.3 (−2.0 to 2.6); 1.3 (−1.1to 3.7); 1.0 (−1.4 to 3.4); (mmol/L) P = .788 P = .272 P = .393

Plasma L-Citrulline

In the statistical analysis of Part A+B, 5 mg apraglutide increasedabsolute plasma L-citrulline by an adjusted mean of 17.6 μmon (95% CI2.0 to 33.2; P=0.031) corresponding to increases of 65% (95% CI 15 to115; P=0.015). In the statistical analysis of Part A, 5 mg apraglutideincreased absolute plasma L-citrulline by an adjusted mean of 17.7 μmon(95% CI −6.3 to 41.7; P=0.100) corresponding to a relative increase of66% (95% CI −13 to 145; P=0.077). The differences in change frombaseline for the 10 mg dose vs placebo were smaller as shown in Table13. In Table 13, the data are shown as an adjusted mean (95% CI),calculations are based on changes from baseline to end of treatment ofindividual dose groups, and N denotes the number of patients in the fullanalysis set. The individual changes from baseline in absoluteconcentration of plasma L-citrulline are plotted in FIG. 11. Mean changeand individual patient plasma L-citrulline changes from baseline areplotted in FIG. 27. Dose-dependent increases in plasma citrulline levelswere observed with apraglutide, starting 2 days after the first dose. Asshown in FIG. 28, following three weekly doses of apraglutide comparedwith placebo, citrulline levels increased significantly.

TABLE 13 Analysis Part A + B Analysis Part A 5 mg vs placebo 10 mg vsplacebo 5 mg vs 10 mg 5 mg vs placebo (N = 8) (N = 8) (N = 8) (N = 8)Plasma 17.6 (2.0 to 33.2); 14.0 (−2.3 to 30.3); −3.6 (−19.8 to 12.6);17.7 (−6.3 to 41.7); L-citrulline P = .031 P = .084 P = .632 P = .100(μmol/L) Relative plasma 65 (15 to 115); 42 (−10 to 94); −23 (−74 to29); 66 (−13 to 145); L-citrulline (%) P = .015 P = .100 P = .344 P =.077

Body Weight and Body Composition

For the analysis of Part A+B, 5 mg apraglutide significantly increasedabsolute and relative fat mass by 1.77 kg (95% CI 0.29 to 3.24; P=0.024)and 10% (95% 1 to 18; P=0.035), respectively. Corresponding to theincrease in fat mass, 5 mg apraglutide increased body weight by 1.9 kg(95% CI 0.1 to 3.7; P=0.043). No changes were found in lean body mass orbone mineral content for any of the dose groups compared to placebo. Inthe statistical analysis of Part A, a trend in increased fat mass andbody weight was observed for 5 mg apraglutide, as shown in Table 14. InTable 14, the data are shown as an adjusted mean (95% CI), calculationsare based on changes from baseline to end of treatment of individualdose groups, and N denotes the number of patients in the full analysisset.

TABLE 14 Analysis Analysis Part A + B Part A 5 mg vs placebo 10 mg vsplacebo 5 mg vs 10 mg 5 mg vs placebo (N = 8) (N = 8) (N = 8) (N = 8)Body 1.9 (0.1 to 3.7); 1.3 (−0.6 to 3.2); −0.6 (−2.4 to 1.3); 1.9 (−0.8to 4.5); weight (kg) P = .043 P = .151 P = .517 P = .128 Relative body 3(0 to 6); 2 (−1 to 5); −1 (−4 to 2); 3 (−1 to 7); weight (%) P = .042 P= .159 P = .487 P = .117 Lean body 0.64 (−2.49 to 3.77); 0.76 (−2.47 to3.99); 0.12 (−3.00 to 3.24); 0.49 (−4.65 to 5.64); mass (kg) P = .656 P= .608 P = .932 P = .721 Relative 2 (−5 to 9); 2 (−6 to 9); −0 (−8 to7); 2 (−11 to 15); lean body P = .521 P = .613 P = .900 P = .547 mass(%) Fat body 1.77 (0.29 to 3.24); 0.33 (−1.20 to 1.85); −1.44 (−2.92 to0.04); 1.74 (−0.36 to 3.83); mass (kg) P = .024 P = .641 P = .055 P =.071 Relative fat 10 (1 to 18); 2 (−7 to 11); −7 (−16 to 1); 9 (−5 to24); body mass (%) P = .035 P = .608 P = .086 P = .110 Bone mineral −4(−73 to 66); 8 (−64 to 81); 12 (−57 to 82); −4 (−132 to 123); content(g) P = .905 P = .799 P = .700 P = .896 Relative 0 (−3 to 3); 0 (−3 to3); 0 (−3 to 3); 0 (−6 to 6); bone mineral P = .916 P = .891 P = .967 P= .927 content (%)

Pharmacokinetics

The plasma concentration of apraglutide increased after dosing, with themaximum mean concentration measured 72 hours after dosing. The meanplasma concentration of apraglutide before the second dose (C_(trough))was 4.5 ng/mL and 8.9 ng/mL in the 5 mg and 10 mg treatment periods,respectively.

Summary of Example 5

The results described in Example 5 demonstrate that the administrationof apraglutide compositions of the present disclosure to subjects wassafe, well tolerated and induced beneficial changes in intestinal fluidabsorption, including, but not limited to, increases in urine volume.

Treatment related adverse events were mild to moderate and correspondedto the physiological effect of GLP-2. There was no dose-dependency foradverse events. Injection site reactions were few, reflecting the justonce weekly dosing regimen. There were no events of treatment relatedabdominal pain or distension.

Treatments with once weekly 5 and 10 mg apraglutide doses increasedurine volume output when compared against placebo. 10 mg apraglutidesignificantly increased urine sodium excretion at end of treatment andreduced oral fluid intake as well as PS volume (assessed near end oftreatment). Similar trends were seen for the 5 mg dose for urine sodiumexcretion and oral fluid intake.

Collectively, these increases in urine volume reflect increasedintestinal fluid and sodium absorption. Without wishing to be bound bytheory, this is an important effect for patients with SBS, who are athigh risk of dehydration, sodium depletion and renal impairment. Fluidand electrolyte abnormalities may also be a cause of morbidity andhospitalizations.

Due to the relatively short treatment period of 4 weeks, reductions inPS volume were made if there were clinical signs of fluid retention. Itis envisioned that during long-term apraglutide treatment, increases inurine output may enable further reductions in PS volume as alsoevidenced by the larger fluid composite effect. Treatment with 5 mgapraglutide increased relative urine volume output in 6 out of 8patients compared to placebo and exceeded the 10% increase which wouldhave triggered a reduction in PS volume.

Plasma concentration of L-citrulline increased during both activetreatment periods. Without wishing to be bound by theory, this indicatesan increased enterocyte mass and is consistent with the intestinotrophiceffects of apraglutide on the intestinal epithelium. In two patients whohad clinically significant increases in urine output (assessed day27-29) in the 5 mg treatment period, a diminished effect of 10 mg wasobserved. However, these patients had concomitant reductions in PSvolume and oral fluid intake (assessed day 20-22) during the 10 mgtreatment period, leading to a positive fluid composite effect, definedas the sum of beneficial effects including increase in urine production,reduction in the need for PS volume and reduction in oral fluid intake.

As would be appreciated by the skilled artisan, patients with SBS maysuffer from secondary hyperaldosteronism due to excessive fecal sodiumlosses and chronic sodium depletion. In the trial described in Example5, it was found that administration of apraglutide tended to decreasealdosterone levels in both dose groups compared to placebo. Withoutwishing to be bound by theory, this indicates that apraglutidealleviates secondary hyperaldosteronism due to improved fluid and sodiumabsorption.

Treatment with 5 mg apraglutide was associated with an increased fatmass and body weight This is the first time an increase in fat mass wasobserved in a subject after treatment with a GLP-2 or GLP-2 analogue t.Increases in body weight correlate well to corresponding changes in fatmass and/or lean mass.

The statistical analysis described in Example 5 was carried out in twoversions, Part A compared 5 mg to placebo, whereas Part A+B includedplacebo, 5 mg and 10 mg to estimate the contrasts between the threetreatments. The results of the Part A and Part A+B analysis weregenerally the same.

In summary, the results described in Example 5 demonstrate that 5 and 10mg of once weekly apraglutide dosing for four weeks was well toleratedand safe. The results also demonstrated clinical effects of a GLP-2analogue with weekly dosing. Once weekly treatment with apraglutideincreased urine volume output and improved other markers of intestinalrehabilitation in patients with SBS at both tested doses (5 and 10 mg)although no differences in response were observed. It is envisioned thatapraglutide compositions of the present disclosure may contribute topatient care and compliance by enabling once weekly, as opposed todaily, administration, which in turn may improve quality of life. Thereduced injection frequency may increase patient acceptability anddecrease the risk of injection site reactions. These results indicatethat apraglutide treatment leads to increased intestinal absorptionthrough promoting intestinal adaptation. Without wishing to be bound bytheory, these results indicate that the treatment with apraglutide invivo can improve intestinal wet weight absorption in a patient withSBS-IF, and therefore can be implemented in a therapeutic setting.

Methods Trial Design and Participants

The trial described in Example 5 was a double-blind, crossover,randomized, placebo-controlled, phase 2 trial followed by an additionaltreatment period in an open-label regimen. Eight adult patients (aged≥18 to ≤80 years) with SBS-IF were enrolled in the trial. Patienteligibility was assessed during a screening visit. The main inclusioncriteria were: Patients with SBS secondary to surgical resection of thesmall bowel with a jejuno- or ileostomy, at least 6 months since lastbowel resection, a fecal output of at least 1500 g/day as recordedwithin the last 18 months, and PS-infusions ≥3 times per week for ≥12months according to the patient's medical record. Patients wereexcluded, if they had clinical signs of activity in inflammatory boweldisease, a history of cancer within ≤5 years or an inadequate hepatic,kidney, or heart function. Patients were also excluded, if they hadreceived native GLP-2 or a GLP-2 analogue within the last 3 months.

As shown in FIG. 4, the trial was organized into two parts: Part A andB. Part A was the double-blind, crossover, randomized,placebo-controlled trial. In Part A, patients were treated with 5 mgapraglutide or placebo once weekly for 4 weeks. After a washout periodof 6-10 weeks, the alternate treatment was given. Part A was followed bya second washout period of 6-10 weeks before patients entered Part B.Part B was an open-label dosing regimen with 10 mg apraglutide givenonce weekly for 4 weeks. The additional open-label treatment period(Part B) was included to investigate the safety and tolerability of 10mg dosing. The procedures during each treatment period were consistent.The trial drug was provided as a freeze-dried powder for reconstitutionand was administered as subcutaneous injections in the abdominal area.

Procedures

Safety assessments were performed for each treatment period and includedobservation for injection site reactions, vital signs, blood samples,electrocardiogram (ECG), dipstick urinalysis, body weight and liverenzymes. They were performed at baseline, during each injection of thetrial drug (pre-dose and at sequential time points), four days afterfirst treatment injection, at the end of each treatment period, and 4-6weeks after the last dosing at the end of the trial, as shown in Table15. Additionally, liver enzymes were measured prior to each drugadministration.

TABLE 15 Local Vital Blood Dipstick Body tolerability signs samples ECGurinalysis weight Baseline visit X X X X X 1st administration ofapraglutide Pre-dose X X X X X 0.5 h after administration X   1 h afteradministration X X X   2 h after administration X X X   3 h afteradministration X   4 h after administration X   6 h after administrationX X X X 4 days after 1st administration of X X X X X apraglutide 2ndadministration of apraglutide Pre-dose X X X X X 0.5 h afteradministration X   1 h after administration X   3 h after administrationX   4 h after administration X X X X End of treatment X X X X X Safetyfollow up (4-6 weeks after X X X X X last treatment at the end of thetrial)

Efficacy assessments were performed for each treatment period of 29days. Patients received a paper diary for recording PS volumeadministration, urine collections and oral fluid intakes at specifictime points. Patients performed home 48-hour urine collections atbaseline (day −2 to 1), immediately after first treatment injection (day1 to 3), and at the end of the treatment period (day 27-29, 5 days afterthe fourth administration of the trial drug). Urine was collected by thepatient in a urine container with volume markings and 48-hour urinevolumes were reported in the diary. After completing the 48-hourcollection, patients transferred approximately 100 mL of urine from thecontainer to a sample which was delivered at the next patient visit. Theamount of sodium excreted per 48 hours was then calculated as theconcentration of sodium in the sample multiplied by the total volume ofurine collected. During each urine collection, weekly PS volume andcontent and daily oral fluid intake were kept constant. Since they werekept constant, increases in urine volume were a sign of increased fluidabsorption. Patients were informed to create a 24-hour drinking menubased on their habitual oral fluid intake at the baseline visit (day−3). Patients were to adhere to their drinking menu during each urinecollection and the concomitant 48-hour oral fluid intake was reported inthe diary during each urine collection.

On day 20-22, patients were informed to record their spontaneous oralfluid intake during 48 hours. This measurement was included toinvestigate whether increased intestinal absorption was associated witha decrease in their spontaneous oral fluid intake.

Between day 4 and 24, PS volume reductions were allowed if deemedclinically necessary by the investigator. Due to the relatively shorttreatment period of 4 weeks, PS volume was only reduced if patients hadclinical signs of fluid retention (such as edema and excessiveunintended weight gain). PS volume reductions were assessed during day20-22. Patients returned to their baseline PS volume and content duringthe end of the treatment urine collection (day 27-29). Concomitantmedications including proton-pump inhibitors, loperamide, and opiateswere kept unchanged and stable throughout the trial. If necessary,adjustments could be made in the drinking menu and prescribed PS volumeand content between treatment periods based on discussions between thepatient and the investigator.

Body weight was measured using a levelled platform scale. Bodycomposition was measured by dual-energy x-ray absorptiometry (NorlandXR-36 DXA densitometer, Ford Atkinson, Wis., USA) at baseline and at endof treatment.

Blood samples for analysis of plasma concentration of apraglutide andfasting plasma L-citrulline, a marker of enterocyte mass, were collectedat baseline, during first and second treatment injection, four daysafter first treatment injection and at the end of treatment. A bloodsample for fasting plasma L-citrulline was also collected 4-6 weeksafter last dosing at the end of the trial. A blood sample foranti-apraglutide antibodies was collected at baseline, at the end oftreatment and 4-6 weeks after last dosing at the end of the trial.

Analysis of Urine Sodium Concentration

Urine sodium was measured with COBAS 8000 modular analyzer series usingan ion-specific electrode system (Roche Diagnostics, Indianapolis, Ind.)with the lower limit of quantification 10 mmol/liter. If urine sodiumwas below the lower limit of quantification, samples were analyzed byflame photometry.

Analysis of Apraglutide Plasma Concentration and L-Citrulline

Apraglutide and L-citrulline were quantified using a validatedLC-MS-based method. For apraglutide, the analytical method usedsolid-phase extraction purification of the intact apraglutide moleculeand its internal standard. The compounds were identified and quantifiedusing reversed-phase HPLC with MS/MS detection using an AB Sciex API5000 quadrupole mass spectrometer. For samples below the limit ofquantification of 5.00 ng/mL, compounds were identified and quantifiedusing reversed-phase UHPLC with MS/MS detection using an AB Sciex API5500 quadrupole mass spectrometer. For L-citrulline, the analyticalmethod used protein precipitation extraction of L-Citrulline and itsinternal standard. The compounds were identified and quantified usingHilic HPLC with MS/MS detection using an AB Sciex API 5000 quadrupolemass spectrometer.

Analysis of Anti-Apraglutide Antibodies

A fully validated ELISA method was used for the detection ofanti-apraglutide antibodies in serum, following a three-tiered assayapproach. In the first tier, anti-apraglutide antibodies present insamples and controls bonded to apraglutide immobilized on a microtiterplate. The bound anti-apraglutide antibodies were then detected withprotein A/G and protein L. A sample with a signal above the validatedscreening cut-point was considered potentially positive and was thentested in the second tier, the confirmatory assay. For this, sampleswere pre-treated with an excess of apraglutide prior to testing them inthe assay described above. An inhibition of the signal equal to orgreater than the validated confirmatory cut-point confirmed the presenceof antibodies. Samples were then reported as anti-apraglutide antibodypositive. Titers were then determined by serial dilution of allconfirmed positive samples.

Statistical Analysis

A crossover design was applied, where each patient served as their owncontrol to eliminate between-subject variability and reduce theinfluence of confounding covariates.

Safety was assessed in all patients who had received at least one doseof trial drug (active or placebo). Efficacy was assessed following amodified intention-to-treat principle including all randomized patientswith at least one valid post-baseline efficacy measurement (fullanalysis set).

Two different statistical analyses were performed: Part A only versionand Part A+B total version. The Part A version was based on a 2×2crossover design, whereas the Part A+B version assumed no period effectin order to estimate the contrasts between the three differenttreatments (placebo, 5 mg, and 10 mg). Both the Part A and Part A+Bversion of the statistical analysis were included in the results. Theanalysis of Part A was randomized and blinded. The analysis of Part A+Bwas included to make the comparison between placebo, 5 mg, and 10 mg. Ananalysis of covariance was used to assess the effects of apraglutide.The period-specific baseline measurement of the outcome variable, oralfluid intake, and PS volume were included in the analysis. Allstatistical tests were done using a two-sided test at a 5% significancelevel. Estimates were presented with approximate 95% confidenceintervals and p-values. SAS version 9.4 was used for the analysis.

Example 6: Phase 2 Metabolic Balance Trial

The following non-limiting example describes results from a Phase 2clinical trial in which subjects with short bowel syndrome intestinalfailure and short bowel syndrome intestinal failure were treated withusing the apraglutide formulations of the present disclosure. This Phase2 trial investigated the safety and efficacy of the apraglutideformulations of the present disclosure.

Treatment with glucagon-like peptide-2 (GLP-2) analogs promotesintestinal adaptation in patients with short bowel syndrome (SBS).Apraglutide is a novel long-acting GLP-2 analog designed to enable onceweekly dosing. This Phase 2 trial investigated the safety and efficacyof once weekly 5 mg apraglutide in patients with SBS intestinal failure(SBS-IF) and SBS intestinal insufficiency (SBS-II). In this open-labeltrial, 8 adult patients with SBS-IF (n=4) or SBS-II (n=4) and a fecalwet weight of ≥1500 g/day were treated with once weekly subcutaneous 5mg apraglutide for 4 weeks. Safety was the primary endpoint. Assecondary endpoints changes were examined from baseline in intestinalabsorption of wet weight, energy (measured by bomb calorimetry) andelectrolytes as well as fecal wet weight and urine production using72-hour metabolic balance studies. Common treatment-related adverseevents were consistent with the physiological effect of GLP-2 andincluded reduced stoma output (n=6), stoma complication (n=6), nausea(n=5), flatulence (n=4), polyuria (n=3) and abdominal pain (n=3). Onceweekly apraglutide significantly increased energy, wet weight andelectrolyte absorption, reduced fecal wet weight and increased urineproduction (Table 16).

TABLE 16 Estimated absolute mean change from baseline (95% CI andp-value, paired t-test) Wet weight absorption (g/day)  741 (194 to 1287;p <0.05) Energy absorption (kJ/day) 1095 (196 to 1994; p <0.05) Fecalwet weight (g/day) −680 (−1200 to −159; p <0.05)   Urine production(g/day)  560 (72 to 1048; p <0.05) Sodium absorption (mmol/day)    38 (3to 74; p <0.05) Potassium absorption (mmol/day)    18 (4 to 32; p <0.05)

Results

Nine patients were screened. One patient did not fulfil the inclusioncriteria of a fecal output ≥1,500 g/day and a urine volume <2,000 mL/day(assessed during the baseline metabolic balance study). Eight patientswere dosed in the trial. All eight patients completed the trial andconstituted the safety and full analysis set, as shown in FIG. 13.Demographics and baseline characteristics of the patients are summarizedin Table 17. Data in Table 17 are mean (SD) or N (%) and parenteralsupport (PS) was scheduled PS at trial entry based on a weekly average.

TABLE 17 SBS-II SBS-IF Total (N = 4) (N = 4) (N = 8) Age (years) 64.5(3.4)  57.5 (20.0) 61.0 (13.8) Sex Female  2 (50%)  3 (75%) 5 (62.5%)Male  2 (50%)  1 (25%) 3 (37.5%) Weight at baseline 83.2 (18.5) 62.5(11.2) 72.8 (18.0) Body-mass index (kg/m2) 28.6 (5.1)  22.7 (4.2)  25.6(5.4)  Race, white 4 (100%) 4 (100%)  8 (100%) Parenteral support volume(mL/day) 2,230 (889)   Parenteral support energy (kJ/day) 2,823 (3,579)Urine production (g/day) 1,423 (212)   1,370 (284)   1,397 (234)  Dietary intake (g/day) 5,710 (1,519) 3,255 (1,006) 4,482 (1,773) Fecaloutput (g/day) 3,419 (2,015) 3,243 (1,339) 3,331 (1,587) Cause ofresection Crohn’s disease  3 (75%) 0 3 (37.5%) Mesenteric vasculardisease  1 (25%)  1 (25%)   2 (25%) Surgical complications 0  3 (75%) 3(37.5%) to ulcerative colitis Disease characteristics Small bowel length(cm) 180 (42)  155 (125) 168 (87)  End-jejunostomy  2 (50%)  2 (50%)   4(50%) lleostomy  2 (50%)  2 (50%)   4 (50%) Colon in continuity 0 0 0Concomitant medication Proton-pump inhibitor 4 (100%)  3 (75%) 7 (87.5%)Opioids or opioid agonists  3 (75%)  3 (75%)   6 (75%) Loperamide  2(50%)  1 (25%) 3 (37.5%)

Safety Results

All patients experienced at least one treatment related adverse event.The adverse events were mild to moderate. Common adverse events aresummarized in Table 18 (data are N or N (%)). Adverse events thatoccurred in >2 patients and included decreased gastrointestinal stomaoutput (n=6), stoma complication (n=6), gastrointestinal stomacomplication (n=5), nausea (n=5), abnormal gastrointestinal stoma output(n=4), flatulence (n=4), polyuria (n=3) and abdominal pain (n=3). Onepatient experienced transient injection site reactions (local erythemaand pruritus) after one injection which was unrelated to the presence ofanti-apraglutide antibodies. Three patients had previously receivednative GLP-2 or another GLP-2 analogue in a clinical trial (minimum of16 months prior to trial inclusion). A total of three serious adverseevents (SAE) occurred in two patients. One SAE, an event of abdominalpain requiring hospital admission, was assessed as related to the trialdrug. The abdominal pain was conservatively treated, and the patient wasdischarged within less than 24 hours. A temporary discontinuation andre-challenge at a reduced dose allowed the patient to complete thetrial. The remaining two SAEs were not considered related toapraglutide. They included one event of acute kidney injury due todehydration in a patient with SBS-II and one event of a CRBSI in apatient with SBS-IF. Two additional patients required a dose reductionin order to complete the trial. One patient with SBS-II had signs offluid retention after the first drug administration, and therefore thesecond and third administrations were given at reduced dose. Thefourth/last administration was given at full dose (5 mg) without furthercomplications. One patient experienced constipation after the first drugadministration, and consequently, the second administration was given atreduced dose. The full dose was reintroduced for the third andfourth/last administration without further complications.

TABLE 18 SBS-II SBS-IF Total (N = 4) (N = 4) (N = 8) Any adverse event 44  8 (100%) Gastrointestinal stoma 3 3   6 (75%) output decreased Stomacomplication 3 3   6 (75%) Gastrointestinal stoma 3 2 5 (62.5%)complication Nausea 1 4 5 (62.5%) Gastrointestinal stoma 3 1   4 (50%)output abnormal Flatulence 3 1   4 (50%) Polyuria 2 1 3 (37.5%)Abdominal pain 1 2 3 (37.5%) Weight increased 2 0   2 (25%) Fluidretention 1 1   2 (25%)

No safety concerns were raised from vital signs, blood samples, ECG,dipstick urinalysis or body weight. One patient with SBS-IF developedanti-apraglutide antibodies during the trial. The antibodies werenegative after a 4-6 week washout. The patient had previously beentreated with a GLP-2 analogue in a clinical trial setting in 2016. Noeffects of anti-apraglutide antibodies were detected on thepharmacokinetic profile, the pharmacodynamic response to apraglutide oron the number or duration of adverse events. None of the patientsdiscontinued the trial due to adverse events and no deaths occurred.

Efficacy Endpoints Wet Weight

The individual changes from baseline following treatment withapraglutide in the wet weight of the dietary intake, fecal output, urineand absorption are shown in FIG. 14. Apraglutide did not change the wetweight of dietary intake, as shown in Table 19. In Table 19, the dataare shown as an adjusted mean (95% CI) and calculations are based onchanges from baseline to end of treatment. Apraglutide s increasedintestinal absorption of wet weight by 741 g/day (95% CI 194 to 1287;P=0.015), as shown in Table 19. Apraglutide significantly decreasedfecal output by 680 g/day (95% CI −1200 to −159; P=0.018) and increasedurine production by 560 g/day (95% CI 72 to 1048; P=0.030), as shown inTable 19.

TABLE 19 Dietary intake Fecal output Urine Absorption Wet weight 61(−84.0 to 207); −680 (−1,200 to −159); 560 (72 to 1,048); 741 (194 to1,287); (g/day) P = .352 P = .018 P = .030 P = .015 Energy 154 (−1,006to 1,314); −941 (−2,438 to 556); — 1,095 (196 to 1,994); (kJ/day) P =.763 P = .181 P = .024 Carbohydrate 154 (−268 to 575); −365 (−772 to43); — 518 (112 to 924); (kJ/day) P = .418 P = .072 P = .019 Lipid 67(−638 to 771); −309 (−969 to 351) — 376 (61 to 691); (kJ/day) P = .830 P= .304 P = .026 Protein −41 (−274 to 191); −145 (−497 to 207); — 104(−205 to 412); (kJ/day) P = .688 P = .362 P = .453 Sodium −5 (−30 to21); −43 (−92 to 6); 27 (5 to 49); 38 (3 to 74); (mmol/day) P = .680 P =.077 P = .024 P = .039 Potassium 4 (−5 to 12); −15 (−32 to 3); 13 (6 to20); 18 (4 to 32); (mmol/day) P = .337 P = .086 P = .003 P = .020Magnesium 1 (−2 to 3); 0 (−9 to 9); 1 (−1 to 2), 0 (−9 to 9); (mmol/day)P = .561 P = .961 P = .411 P = .930 Calcium −2 (−5 to 2); −13 (−36 to11); 0 (−1 to 1) 11 (−13 to 35); (mmol/day) P = .367 P = .255 P = .419 P= .313

Electrolytes

Apraglutide increased absorption of sodium and potassium by 38 mmol/day(95% CI 3 to 74; P=0.039) and 18 mmol/day (95% CI 4 to 32; P=0.020)respectively, as shown in Table 19. Urine sodium and potassium excretionincreased by 27 mmol/day (5 to 49; P=0.024) and 13 mmol/day (95% CI 6 to20; P=0.003) respectively, as shown in Table 19. There was no change inthe absorption or urine excretion of magnesium and calcium, as shown inTable 19. The electrolyte content of dietary intake and fecal output didnot change, as shown in Table 19. Individual changes from baseline inelectrolyte absorption are shown in FIG. 15.

Energy and Macronutrients

FIG. 16 shows the individual changes from baseline in the energy contentof dietary intake, fecal output and absorption. Apraglutide did notchange total dietary energy intake or any individual macronutrient, asshown in Table 19. Compared to baseline, apraglutide increasedintestinal absorption of energy by 1,095 kJ/day (95% CI 196 to 1,994;P=0.024), as shown in Table 19. With a suggestion of improvements inenergy absorption, the energy content of fecal output decreased by 941kJ/day (95% CI −2,438 to 556 P=0.181), as shown in Table 19.Carbohydrate and lipid absorption significantly increased by 518 kJ/day(95% CI 112 to 924; P=0.019) and 376 kJ/day (95% CI 61 to 691; P=0.026)respectively, as shown in Table 19. Protein absorption increased by 104kJ/day (95% CI −205 to 412; P=0.453), as shown in Table 19. Theindividual changes in macronutrient absorption are plotted in FIG. 17.

Body Weight and Body Composition

Body weight increased by 1.8 kg (95% CI 0.4 to 3.1; P=0.016) after fourweeks of apraglutide treatment. Lean body mass significantly increasedby 1.7 kg (95% CI 0.8 to 2.6; P=0.003) and fat mass decreased by 1.1 kg(95% CI −2.1 to −0.0; P=0.044). There was no significant change in bonemineral content which changed by −20 g (95% CI −58 to 19; P=0.268).

Citrulline

Compared to baseline, apraglutide increased absolute and relative plasmaconcentration of L-citrulline by 15.2 μmon (95% CI 3.3 to 27.1; P=0.019)and 66% (95% CI 3 to 128; P=0.043), respectively.

Pharmacokinetics

The plasma concentration of apraglutide increased rapidly after dosing,with maximum mean concentration of 118.0 ng/mL reached after a mean of29.5 hours. The mean elimination half-life was 27.0 hours and meanclearance was 0.9 L/h. W

Summary of Example 6

The results described in Example 6 demonstrate that 5 mg apraglutideadministered once weekly was safe and well tolerated in patients withSBS-II and SBS-IF after four weeks of treatment and showed positiveeffects on intestinal absorption, which in a clinical setting, wouldeliminate or reduce the need for PS.

Adverse events were consistent with the known physiological effects ofGLP-2. Frequently reported related adverse events were ofgastrointestinal origin, but they were generally mild and transient.

Apraglutide significantly increased intestinal absorption of wet weight,energy and electrolytes (sodium and potassium). The improvements inabsorption were accompanied by a significant decrease in fecal wetweight and increase in urine production and urine electrolyte excretion(sodium and potassium). The changes in urine production found in thistrial are considered clinically relevant as they could enable PSreductions or help regain enteral autonomy in patients with SBS-IF. Itis envisioned that these improvements could eventually also reduce therisk of developing intermittent or chronic IF in patients with SBS-IIand alleviate the symptom burden of malabsorption.

The pharmacokinetic profile of apraglutide supported a once weeklydosing regimen.

Apraglutide is the first GLP-2 analogue to improve absorption of energyacross the whole patient spectrum in the SBS population when measured bybomb calorimetry, which is regarded as the gold standard laboratorymethod for quantifying intestinal energy absorption. For individualmacronutrients, apraglutide increased absorption of carbohydrates andlipids, whereas protein absorption remained unchanged. As would beappreciated by the skilled artisan, in later stages of clinicaldevelopment, weaning off PS with decreasing energy requirements isconsidered evidence of an improved energy absorption.

The PK profile of apraglutide allowed for consistent exposure whichmight explain the improved effects on energy absorption.

Apraglutide significantly increased body weight and lean body mass andreduced fat mass, which, as would be appreciated by the skilled artisan,indicates possible improvements in hydration status.

Apraglutide did not change the wet weight or energy content of dietaryintake. Although oral fluid intake was fixed, the dietary intake ofsolids was unrestricted. GLP-2 did not significantly affect appetite orpostprandial feeling of satiety in healthy humans. A reduced dietaryintake could be an undesired side-effect in patients with SBS who dependon hyperphagia to compensate for the intestinal losses. Long-term GLP-2treatment allowed patients to reduce their dietary intake whilemaintaining the same degree of absorption. Thus, it is envisioned thatadministration of apraglutide might alleviate the need for severehyperphagia which is a predominant symptom in especially patients withsevere SBS-II.

Apraglutide significantly increased plasma L-citrulline, a marker ofenterocyte mass, providing support for its expected pro-adaptiveeffects. GLP-2 also inhibited gastrointestinal motility and reducedgastric acid secretion and thereby could increase exposure of theenterocyte to the luminal content. Mesenteric blood flow can also bestimulated by GLP-2, which could increase nutrient absorption. GLP-2 mayalso upregulate transport proteins as shown in animal studies.

The results described in Example 6 indicate that 5 mg apraglutidetreatment administered weekly for four weeks is safe and well toleratedin patients with SBS-IF and SBS-II. These results demonstrate for thefirst time, a once weekly GLP-2 analog significantly improved absorptionof wet weight, energy, electrolytes and increased urine production.Apraglutide only requires weekly injections which may reduce theinjection burden and contribute to patient care and compliance. Thereduced injection frequency may also increase patient acceptability anddecrease the risk of injection site reactions.

Once weekly treatment with apraglutide increased intestinal absorptionof fluid, electrolytes and energy. Without wishing to be bound bytheory, these results indicate that the treatment of patients with theapraglutide compositions of the present disclosure can improveintestinal wet weight absorption and energy in a patient with SBS-IF,and therefore can be implemented in a therapeutic setting.

Methods Trial Design and Participants

A total of nine adult patients (aged ≥18 to ≤80 years) with SBS werescreened, eight of whom enrolled in the trial: four patients had SBS-IIand four patients had SBS-IF. Both subgroups of patients were includedto investigate the safety and efficacy of apraglutide across the diseasespectrum. Patient eligibility was assessed during a screening visit.Main inclusion criteria were SBS secondary to surgical resection of thesmall intestine with or without colon; at least six months since lastsurgical bowel resection; and a severe degree of malabsorption, definedas a fecal wet weight output ≥1,500 g/day and a urine volume production<2000 mL/day. Fecal output and urine volume production were confirmedduring baseline examinations. Patients were excluded if they hadclinical signs of active inflammatory bowel disease, a history of cancerwithin five years or an inadequate hepatic-, kidney- or heart function.Patients were also excluded if they were pregnant or breastfeeding, hada positive HIV, hepatitis B or C test, had been hospitalized within onemonth before the screening visit or had received native GLP-2 or GLP-2analogue within the last three months.

Procedures

Patients were treated with once weekly 5 mg apraglutide for four weeks.Apraglutide was provided as a freeze-dried powder for reconstitution insterile water prior to injection, and treatments were administered assubcutaneous injections in the abdominal area. A 72-hour metabolicbalance study was performed at baseline and at the end of the treatmentperiod (starting one day after the fourth and last apraglutideinjection). Each 72-hour metabolic balance study was performed during afive-day hospital admission. On the day of admission, patients wereinstructed to create a 24-hour drinking menu based on their habitualoral fluid intake. The drinking menu was to be followed during eachbalance study. On the second day of admission, just after the patientshad urinated and emptied their stoma bags or defecated, the balancestudy was initiated. Patients were instructed to collect their fecaloutput, urine and a precise duplicate of their dietary intake (fluidsand solids separated) in respective buckets which were replaced after 24hours. Patients had free access to food but daily PS (volume andcontent) and oral fluid intake were kept constant during the baselineand post-treatment metabolic balance study periods. Daily PS and oralfluid intake were kept constant to ensure that the baseline andpost-treatment measurements were comparable with regards to measuringthe treatment effect. Daily PS and compliance to the predefined drinkingmenu was documented during admissions. Concomitant medications includingproton pump inhibitors, loperamide and opiates remained unchangedthroughout the trial.

The contents of the buckets were weighed, processed intro dry matter,and analyzed as previously described: energy by bomb calorimetry,nitrogen by Kjeldahl's method, lipid by a modified Van de Kamertitration technique, carbohydrate by Englyst's method, sodium andpotassium by flame photometry and calcium and magnesium by atomicabsorption spectrometry. A 24-hour average was calculated based on thethree 24-hour periods. Absolute changes were calculated as thedifference between the baseline and the post-treatment value. Relativechanges were calculated as absolute changes divided by baseline valuesmultiplied by 100.

Body weight was measured using a levelled platform scale. Bodycomposition was measured by dual-energy x-ray absorptiometry (NorlandXR-36 DXA densitometer, Norland, Ford Atkinson, Wis., USA) at baselineand post-treatment.

First, second and fourth apraglutide injections were performed at thehospital. Safety assessments included observation for injection sitereactions, vital signs, blood samples, electrocardiogram (ECG),urinalysis and body weight. They were performed at baseline, during eachinjection of the trial drug, four days after first injection, at the endof treatment and 4-6 weeks after last dosing at the end of the trial, asshown in Table 20. Liver enzymes were measured prior to each drugadministration.

TABLE 20 Local Vital Blood Dipstick Body tolerability signs samples ECGurinalysis weight Baseline visit X X X X 1st administration ofapraglutide Pre-dose X X X X X 0.5 h after administration X   1 h afteradministration X X X   2 h after administration X X   3 h afteradministration X   4 h after administration X   6 h after administrationX X X 4 days after 1st administration of X X X apraglutide 2ndadministration of apraglutide Pre-dose X X X X 0.5 h afteradministration X   1 h after administration X   2 h after administrationX   3 h after administration X X X X   4 h after administration 4thadministration of apraglutide Pre-dose X X X 0.5 h after administrationX   1 h after administration X   3 h after administration X End oftreatment X X X Safety follow up (4-6 weeks after last treatment at theend of the X X X X trial)

Blood samples for analysis of the plasma concentration of apraglutideand plasma concentration of fasting L-citrulline, a marker of enterocytemass, were collected at baseline, during first and second injection(pre-dose and at sequential timepoints), four days after first injectionand at the end of treatment. Blood sampling for apraglutidepharmacokinetics was also performed after the fourth and last injectioncovering 96 hours. A blood sample for fasting plasma L-citrulline wasalso collected 4-6 weeks after last dosing at the end of the trial.Blood samples for anti-apraglutide antibodies were collected atbaseline, at the end of treatment and at the 4-6 weeks follow-up visit.All eight patients tested negative for anti-apraglutide antibodies atbaseline.

Outcomes

The primary endpoint of this trial was safety. Secondary endpoints wereabsolute and relative changes from baseline in dietary intake, fecalexcretion and absorption of wet weight, energy, macronutrients andelectrolytes, urine production, urine electrolyte excretion, bodyweight, body composition, bone mineral content, plasma L-citrulline aswell as pharmacokinetic profiles. Only absolute changes were presentedin the scope of this disclosure with the exception of changes frombaseline in plasma L-citrulline.

Analysis of Plasma Concentration of Apraglutide and L-Citrulline

Apraglutide and L-citrulline were quantified using a validatedLC-MS-based method. For a apraglutide, the analytical method usedsolid-phase extraction purification of the intact apraglutide moleculeand its internal standard. The compounds were identified and quantifiedusing reversed-phase HPLC with MS/MS detection using an AB Sciex API5000 quadrupole mass spectrometer. For samples below the limit ofquantification of 5.00 ng/mL, compounds were identified and quantifiedusing reversed-phase UHPLC with MS/MS detection using an AB Sciex API5500 quadrupole mass spectrometer. For L-citrulline, the analyticalmethod used protein precipitation extraction of L-citrulline and itsinternal standard. The compounds were identified and quantified usingHilic HPLC with MS/MS detection using an AB Sciex API 5000 quadrupolemass spectrometer.

Analysis of Anti-Apraglutide Antibodies

A fully validated ELISA method was used for the detection ofanti-apraglutide antibodies in serum, following a three-tiered assayapproach. In the first tier, anti-apraglutide antibodies present insamples and controls bonded to apraglutide immobilized on a microtiterplate. The bound anti-apraglutide antibodies were then detected withprotein A/G and protein L. A sample with a signal above the validatedscreening cut-point was considered potentially positive and was thentested in the second tier, the confirmatory assay. For this, sampleswere pre-treated with an excess of apraglutide prior to testing them inthe assay described above. An inhibition of the signal equal to orgreater than the validated confirmatory cut-point confirmed the presenceof antibodies. Samples were then reported as anti-apraglutide antibodypositive. Titers were then determined by serial dilution of allconfirmed positive samples.

Statistical Analysis

A statistical test of adjusted mean change from baseline to end oftreatment was analyzed using a paired t-test. All statistical tests weredone using a two-sided test at a 5% significance level. Estimates werepresented with approximate 95% confidence intervals and p-values. SASversion 9.4 was used for the analysis.

Example 7: Pharmacokinetic/Pharmacodynamic Evaluation Demographics andOther Baseline Characteristics

11 females and 13 males were included in the study. The demographic andbaseline data are summarized in Table 21. No relevant medical historywas reported. The most frequent reported prior concomitant medicationwas regarding birth control. Following first dosing, the most frequentreported concomitant medication was paracetamol. No relevant differencesin concomitant medications were noted between the treatment groups.

TABLE 21 1 mg 5 mg 10 mg apraglutide apraglutide apraglutide PlaceboTotal (N = 6) (N = 6) (N = 6) (N = 6) (N = 24) Number 3/3 3/3 2/4 3/311/13 of female/ male subjects Age in years 26.7 ± 6.3  24.5 ± 4.0  27.7± 7.8  29.2 ± 7.5  27.0 ± 6.4  (mean ± SD) Height in cm 178.02 ± 8.11 178.52 ± 11.39  175.48 ± 8.29  175.67 ± 11.19  176.92 ± 9.31  (mean ±SD) Weight in kg 75.683 ± 12.544 67.550 ± 11.070 72.150 ± 14.237 71.233± 12.382 71.654 ± 12.123 (mean ± SD) BMI in kg/m² 23.72 ± 2.02  21.13 ±2.41  23.22 ± 2.22  22.98 ± 2.30  22.76 ± 2.32  (mean ± SD) Number of0/0/1/5 1/1/0/4 0/1/0/5 0/0/0/6 1/2/1/20 American Indian or AlaskaNative/ Asian/Mixed / White subjects BMI = body mass index; SD =standard deviation. Measurements of Treatment Compliance

Treatments were subcutaneously (SC) administered to the subjects bymembers of clinical staff, there was full treatment compliance.

Pharmacodynamic and Efficacy Results

The plasma citrulline concentrations against time after first dose ofapraglutide per dose level are presented in FIG. 18. The correspondingleast square (LS) mean plot for change from baseline is provided in FIG.19. Summary pharmacokinetic parameters of citrulline are provided inTable 22.

The endogenous marker citrulline was selected to assess thepharmacodynamic effect of apraglutide. Citrulline was detected in allbaseline and post-dose samples. Three baseline citrulline samples wereobtained during two baseline visits between screening and the firstdosing day and before dosing on the first dosing day. Thirteen out of 24subjects had a difference of <0.5 μg/mL over the three baseline sampleswith the smallest difference being 0.061 μg/mL observed for a 10 mgdosed subject. Another five and four subjects had a difference between0.5-1 and 1-2 μg/mL across baseline assessments, respectively. Theremaining two subjects had a difference >2 μg/mL with the largestdifference being 2.762 μg/mL observed for a 1 mg dosed subject. Atbaseline, the mean citrulline range was 5.3825-5.6207 μg/mL for placebo,5.3900-6.2028 μg/mL for 1 mg apraglutide, 4.8983-5.3023 μg/mL for 5 mgapraglutide and 5.0730-5.4690 μg/mL for 10 mg apraglutide. In theplacebo group, the mean citrulline concentration remained relativelystable over the trial, ranging from 4.9735 to 5.8253 μg/mL.

Following the first weekly SC dose of apraglutide, citrulline wasincreased dose-dependently with the highest citrulline levels measuredafter four or seven days, with the exception of one subject in the 1 mggroup whose maximum citrulline concentration was reached after two days.The highest mean citrulline value was 6.3920 μg/mL (after four days),7.1933 μg/mL (after four days) and 7.8487 μg/mL (after 7 days), for 1, 5and 10 mg, respectively, while for placebo it was 5.8325 μg/mL (afterone day).

The concentration at the end of each dosing interval, R_(pre-dose), wasassessed for each dose and showed that in general, citrulline levelswere similar over the whole treatment period (Table 22).

Following the sixth and last weekly SC dose of apraglutide, an increasewas observed at all dose levels and, in particular, for the highestdoses. The mean R_(max) was 7.1702, 8.1577 and 8.7254 μg/mL reached at2.0, 3.9 and 3.9 days following the last dose of 1, 5 and 10 mg,respectively (Table 22). The corresponding placebo level was 6.3707μg/mL at 16.9 days following the last dose. These R_(max) values wereonly slightly lower than the mean R_(max) assessed over the whole trial,with differences ranging from 0.0362, 0.0973 and 0.1016 μg/mL for 10, 5and 1 mg, respectively. For placebo, the difference was 0.2586 μg/mL.The mean R_(max) occurred around the same time for all apraglutidegroups, ranging from 35.9 to 38.9 days following the first dose of 1 and5 mg, respectively, while placebo peaked at 50.4 days following thefirst dose.

The mean citrulline increase observed following the last dose appearedto decline more rapidly for the lowest doses compared to 10 mg.Citrulline was assessed up to day 77. The lowest mean citrulline levelfollowing the last dose was reached 17 days later for both 1 and 10 mg.For 5 mg the lowest mean citrulline level was reached after anadditional week. Rt_(1/2) was assessed for two subjects, both dosed with10 mg, and was 6.5 and 11.0 days (Table 22).

TABLE 22 Apraglutide dose level Placebo 1 mg 5 mg 10 mg Last doseArithmetic mean ± standard deviation 1 R_(pre-dose) (μg/mL) 5.2028 ±1.1888 5.8075 ± 1.3328 6.7695 ± 1.5288 7.8487 ± 2.7273 2 R_(pre-dose)(μg/mL) 5.1740 ± 1.0442 6.2605 ± 1.2331 7.0082 ± 1.1330 7.6058 ± 2.32103 R_(pre-dose) (μg/mL) 4.9735 ± 1.3734 6.4913 ± 1.8297 6.6155 ± 1.27907.5180 ± 2.1369 4 R_(pre-dose) (μg/mL) 5.4545 ± 0.8906 6.0050 ± 1.57906.6227 ± 1.2247  7.2060 ± 2.5488* 5 R_(pre-dose) (μg/mL) 5.4278 ± 1.31306.4128 ± 1.8032 6.8307 ± 1.2369  7.1994 ± 2.5111* 6 R_(max) (μg/mL)6.3707 ± 1.6653 7.1702 ± 1.6611 8.1577 ± 1.7108  8.7254 ± 3.0145*R_(pre-dose) (μg/mL) 5.2883 ± 1.5281 6.3120 ± 1.6694 7.2498 ± 1.2461 8.0714 ± 3.0514* NA R_(max) (μg/mL) 6.6293 ± 1.4010 7.2718 ± 1.69598.2550 ± 1.7807  8.7616 ± 2.9900* Median (minimum, maximum) 6 Rt_(max)(d) 16.9 (0.9, 41.9)  2.0 (0.0, 34.9) 3.9 (0.0, 6.9) 3.9 (1.9, 7.0)* Rt½( d) ND ND ND  8.8 (6.5, 11.0)** NA Rt_(max) (d) 50.4 (0.9, 77.0) 35.9(0.0, 70.0) 38.9 (4.0, 41.9) 36.9 (7.0, 39.0)* Geometric mean (geometriccoefficient of variation %) 1 R_(pre-dose) (μg/mL) 5.0803 (24.9) 5.6723(24.6) 6.5967 (26.7) 7.4048 (40.7) 2 R_(pre-dose) (μg/mL) 5.0802 (21.7)6.1526 (21.0) 6.9208 (18.1) 7.2438 (37.6) 3 R_(pre-dose) (μg/mL) 4.8114(29.1) 6.2752 (29.2) 6.5109 (19.9) 7.2331 (32.4) 4 R_(pre-dose) (μg/mL)5.3963 (16.0) 5.8213 (28.4) 6.5313 (18.3)  6.8103 (40.3)* 5 R_(pre-dose)(μg/mL) 5.2867 (26.1) 6.2097 (28.2) 6.7248 (20.3)  6.7908 (41.8)* 6R_(max) (μg/mL) 6.1904 (26.7) 7.0069 (24.0) 8.0024 (22.0)  8.2533(40.4)* R_(pre-dose) (μg/mL) 5.0890 (31.9) 6.1166 (28.5) 7.1475 (19.3) 7.5618 (44.0)* NA R_(max) (μg/mL) 6.5079 (21.3) 7.1036 (24.2) 8.0870(22.9)  8.2953 (40.1)* NA = not applicable, assessed over the wholetrial period; ND = no data available; R_(max) = maximum responseR_(Pre-dose) = response immediately prior to dosing; Rt_(max) = time toreach maximum response; Rt½ = response half-life. Number of subjects (N)was 6 unless indicated otherwise. *1 subject missing (N = 5) and **4subjects missing (N = 2).

The variability across R_(pre-dose) and R_(max) (both following the lastdose and throughout the trial) expressed as coefficient of variation (%CV) ranged for placebo from 16.0 to 31.9% (Table 22). This was in linewith the % CV range observed for 1 and 5 mg of 18.1 to 29.2%. The % CVfor 10 mg was slightly higher, 32.4 to 44.0%.

A statistical steady-state analysis per dose level was performed usingHelmert's approach. In short, the first contrast tested compared themean R_(pre-dose) at the first time point (week 1) to the pooled meanR_(pre-dose) over all remaining time points (week 2 to 6). The secondcontrast compared the mean at week 2 to the pooled mean over week 3 to6, etc. Testing continued until the contrast was not statisticallysignificant. The mean R_(pre-dose) at week 6 for 10 mg against meanR_(pre-dose) over week 5 (0.8720, 95% CI: 0.1590; 1.5850 and p=0.0172)was statistically significant. Visual inspection of the data suggeststhat steady state concentrations were reached following the first dosingat all investigated dose levels.

The citrulline data were analyzed using a mixed model analysis ofvariance. A summary of the analysis results is provided in Table 23. Thestatistical analysis clearly showed a significant overall treatmenteffect of apraglutide on citrulline (p=0.0007). All dose groups showedan increase compared to placebo, although the difference between placeboand 1 mg was not statistically significant. The increase compared toplacebo became more pronounced and significant with increasing dose,namely an increase of 1.2574 μg/mL for the 5 mg dose (p=0.0025) and1.6343 μg/mL for 10 mg (p=0.0002) (Table 23). In addition, whencomparing the dose levels to each other, the 5 and 10 mg doses inducedsignificantly higher citrulline levels compared to the 1 mg dose (0.9429μg/mL higher [p=0.0186] and 1.3198 μg/mL higher [p=0.0018],respectively). The difference between the 5 and 10 mg doses was notstatistically significant, although the effect following 10 mg appearedto be slightly higher than after 5 mg.

TABLE 23 Treatment Placebo- Placebo- Placebo- 1 mg- 1 mg- 5 mg-Parameter p-value 1 mg 5 mg 10 mg 5 mg 10 mg 10 mg Citrulline 0.00070.3146 1.2574 1.6343 0.9429 1.3198 0.3769 (μg/mL) (−0.4371, (0.5037,(0.8809, (0.1768, (0.5586, (−0.3766, 1.0663) 2.0112) 2.3878) 1.7089)2.0810) 1.1304) P = 0.3910 P = 0.0025 P = 0.0002 P = 0.0186 P = 0.0018 P= 0.3078 Bristol stool 0.0189 −0.9 −1.0 −0.6 −0.1 0.3 0.5 scale (−1.6,−.3) (−1.7, −.4) (−1.3, 0.1) (−0.8, 0.6) (−0.3, 1.0) (−0.2, 1.1) P =0.0092 P = 0.0046 P = 0.0961 P = 0.7531 P = 0.3020 P = 0.1928 Bristolstool 0.3031 −0.6 −0.4 −0.5 0.2 0.1 −0.0 scale with (−1.3, 0.1) (−1.1,0.3) (−1.2, 0.2) (−0.5, 0.9) (−0.6, 0.8) (−0.8, 0.7) zeroes set to P =0.0808 P = 0.2018 P = 0.1648 P = 0.6208 P = 0.7282 P = 0.8876 missingWeight (kg) 0.7104 −0.750 −0.865 −0.331 −0.115 0.419 0.534 (−2.497,(−2.598, (−2.065, (−1.896, (−1.324, (−1.215, 0.997) 0.868) 1.402) 1.666)2.162) 2.283) P = 0.3800 P = 0.3092 P = 0.6941 P = 0.8939 P = 0.6210 P =0.5309 The overall treatment p-value (all dose groups compared toplacebo) and subsequent treatment group comparisons are shown. Estimatesof the difference with 95% confidence intervals and p-values are shownper comparison.

Besides citrulline, the Bristol stool scale and body weight wereassessed as pharmacodynamic endpoints. In contrast to citrulline andweight, the Bristol stool scale is a discrete variable. Subjectsassessed their stool in the past 24 hours using the Bristol stool scaleranging from constipation (type 1) to diarrhea (type 7). No stool in thepast 24 hours was recorded as zero. As two subjects (assigned to either1 or 5 mg) reported no stool preceding the first dose, the screeningBristol stool scale score was also taken into account when determiningthe baseline Bristol stool scale score.

A significant overall treatment effect of apraglutide on the Bristolstool scale was observed, with a p-value of 0.0189 (Table 23). Thespecific placebo versus dose level contrasts showed a significantdecrease in the 1 and 5 mg groups compared to placebo (−0.9, p=0.0092and −1.0, p=0.0046); the decrease in the 10 mg group compared to placebowas not statistically significant. This pattern appeared to be driven bya few subjects who occasionally reported no stool. Following the firstdose, assessment of the Bristol stool scale was scheduled at 11 visitsand at trial discharge. Of these visits, no stool in the past 24 hourswas reported 6 times (2 subjects), 13 times (4 subjects) and 1 time (1subject) in the 1 mg, 5 mg, 10 mg group, respectively. In the placebogroup, all subjects reported stool in the past 24 hours at each visit.No stool was reported 9 times during the 6 dosing weeks and 11 times inthe subsequent 6 weeks, indicating no association with dosing.

Considering the potential impact of scoring no stool as 0 when a subjectmay not have normally defecated every 24 hours (supported by theobservation that 2 out of 24 subjects reported 0 prior to the firstdose), the analysis was repeated as a sensitivity analysis with no stoolset to missing. With this adjusted analysis, no significant treatmenteffect was found either overall or for any of the specific treatmentcontrasts (Table 23).

Body weight appeared to be rather constant over the whole trial period,with no statistically significant treatment effect of apraglutide onbody weight overall or for any of the specific treatment contrasts(Table 23). When considering individual body weight values, placebosubject 1007 showed a weight increase from 92.90 kg at screening to99.30 kg at trial discharge visit.

Citrulline, BSS and weight data are analyzed with a mixed model analysisof variance with fixed factors treatment, time and treatment by time,random factor subject, and the average prevalue as covariate. Theaverage pre-value was calculated from all pre-values after screening andbefore dosing, except for BSS, where the screening value was also usedto calculate the average baseline.

MCP-MOD Analysis

The MCP-MOD procedure was applied. This statistical methodologyconsisted of 2 steps: the multiple comparisons step and the modellingstep. The first step of the procedure (MCP) is used to test for asignificant dose response by assessing pre-specified candidate models.Once a dose response has been established, the second step (MOD) is usedto estimate a dose-response curve and estimation of target doses ofinterest. Five candidate models were specified:

-   -   Linear model with maximum effect for the 10 mg dose    -   Log-linear model with maximum effect for the 10 mg dose    -   Dose that induces maximum effect (E_(max)) model with ED₅₀ of 5        mg    -   Sigmoidal E_(max) model with ED₅₀ of 1 mg and hill coefficient        (HILL) of 2    -   Sigmoidal E_(max) model with ED₅₀ of 5 mg and HILL of 5

The results of the MCP step showed that all 5 candidate dose responsemodels generated significant contrasts with a p-value of <0.0001 (Table24). The estimates of the differences were slightly distinctive andresulted in a set of models with a better fit and a set with slightlyworse fit. The candidate dose response models yielding the largestestimate of the difference were the 1) E_(max) model with ED₅₀=5 mg, 2)log-linear model and 3) sigmoidal E_(max) model with ED₅₀ of 1 mg andHILL of 2 (Table 24). As these estimates of the difference were verysimilar, all 3 models were taken into the MOD part. The models arereferred to as 1) E_(max), 2) Log Lin and 3) Sigmoid E_(max) model,respectively.

TABLE 24 Estimate Candidate dose Test of the 95% CI response modelstatistic p-value difference Lower Upper Linear 5.704 <0.0001 2.07811.3127 2.8435 Log-linear 6.168 <0.0001 2.2125 1.4588 2.9661 E_(max) withED₅₀ = 5 mg 6.172 <0.0001 2.2150 1.4610 2.9689 Sigmoid E_(max) with6.144 <0.0001 2.1663 1.4256 2.9071 ED₅₀ = 1 mg and H = 2 Sigmoid E_(max)with 5.687 <0.0001 2.0713 1.3061 2.8365 ED₅₀ = 5 mg and H = 5 CI =confidence interval; ED₅₀ = dose that induces 50% of maximum effect;E_(max) = maximum effect; H = hill coefficient; MCP-MOD = multiplecomparison procedure-modelling. Doses comprised of 0 (placebo), 1, 5 and10 mg apraglutide. Citrulline response data comprised the averagebaseline value and day 35 to 42 (= week 6) data. Per the candidate doseresponse model, the analysis result is characterized by test statistic,p-value of the contrast and estimate of the difference with 95% CI.

A subset of the citrulline data was made with the average pre-value andday 35 to 42=week 6 data. The coefficients were implemented in the mixedmodel analysis of variance with fixed factors treatment, time andtreatment by time, random factor subject, and the average prevalue ascovariate. The average pre-value was calculated from all pre-valuesafter screening and before dosing.

The following contrasts are calculated within the model:

-   -   Linear with coefficients −0.508 −0.381 0.127 0.762    -   LinLog with coefficients −0.654 −0.283 0.306 0.631    -   Emax with coefficients −0.632 −0.316 0.316 0.632    -   Sigmoid Emax Hill=2 ED50=1 with coefficients −0.759 −0.140 0.432        0.467    -   Sigmoid Emax Hill=5 ED50=5 with coefficients −0.456 −0.455 0.164        0.747

The MOD step showed that the predicted citrulline change from placebofor a dose of 1, 5 and 10 mg were rather similar for all 3 models (Table25). With a dose of 1 mg, the predicted placebo and baseline correctedchange in citrulline across the 3 models ranged from 0.7125 to 0.8712μg/mL with significant results for the E_(max) (p=0.0184) and Log-linear(p=0.0238) models. For the Sigmoid E_(max) model, the p-value was0.0645. When increasing the dose to 5 mg, the predicted change fromplacebo increased to a range of 2.1652 to 2.3327 μg/mL. For a dose of 10mg, the predicted change from placebo was slightly higher and rangedfrom 2.6946 to 2.8604 μg/mL. The predicted response versus placebo forboth 5 and 10 mg was statistically significant for all models, with ap-value of <0.0001.

The apraglutide dose levels required for a baseline and placebocorrected citrulline increase of 1, 2 and 3 μg/mL were predicted (Table25). The E_(max) of the Sigmoid E_(max) model was <3 μg/mL, so for thismodel a change of 2.5 μg/mL was chosen as largest response. Thepredicted apraglutide dose for a citrulline increase of 1 μg/mL was1.2333 mg, 1.2249 mg and 1.3606 mg, for the E_(max), Log Lin and SigmoidE_(max) model, respectively. To evoke a citrulline increase of 2 μg/mL,the predicted apraglutide doses were 3.8471 mg, 4.2011 mg and 3.4042 mgfor the E_(max), Log Lin and Sigmoid E_(max) model, respectively.Finally, the predicted apraglutide doses for change from placebo of 2.5or 3 μg/mL were 13.1053 mg, 11.4325 mg and 6.4619 mg using the E_(max),Log Lin and Sigmoid E_(max) model, respectively. It should be noted thatthe uncertainty in the predicted apraglutide doses is rather large,especially to achieve an effect of 2.5 or 3 μg/mL.

TABLE 25 Prediction E_(max) Log-linear Sigmoid E_(max) Predicted changefrom placebo 0.8535 0.8712 0.7125 effect for dose of 1 mg (0.11584,1.5486) (0.1273, 1.6151) (−0.0466, 11.4716) p = 0.0184 p = 0.0238 p =0.0645 Predicted change from placebo 2.2442 2.1652 2.3327 effect fordose of 5 mg (1.4239, 3.0644) (1.3805, 2.9499) (1.5737, 3.0916)  p =<0.0001  p = <0.0001  p = <0.0001 Predicted change from placebo 2.81822.8604 2.6946 effect for dose of 10 mg (1.9820, 3.6544) (2.0011, 3.7197)(1.8761, 3.5131)  p = <0.0001  p = <0.0001  p = <0.0001 Predicted dosefor change from 1.2333 1.2249 1.3606 placebo of 1 μg/mL (−0.04246,2.5091) (−0.2169, 2.6667) (0.2680, 2.4532) p = 0.0574 p = 0.0920 p =0.0170 Predicted dose for change from 3.8471 4.2011 3.4042 placebo of 2μg/mL (0.3534, 7.3409) (0.6406, 7.7617) (−0.1290, 6.9374) p = 0.0324 p =0.0229 p = 0.0582 Predicted dose for change from ND ND 6.4619 placebo of2.5 μg/mL* (−0.5521, 13.4759) p = 0.0692 Predicted dose for change from13.1053 11.4325 ND placebo of 3 μg/mL* (−6.3068, 32.5173) (1.5808,21.2843) p = 0.1754 p = 0.0249 CI = confidence interval; E_(max) =maximum effect; ND = not determined. *The Emax of the Sigmoid Emax modelwas <3 μg/mL, therefore the largest response was set at 2.5 μg/mL.Predictions were made based on doses of 0 (placebo), 1, 5 and 10 mgapraglutide and citrulline response data comprised of the averagebaseline value and day 35 to 42 (= week 6) data. Estimated change/dosewith 95% CIs and p-values are shown per prediction.

Adjustments for Covariates

For all pharmacodynamic endpoints (citrulline, Bristol stool scale andweight), the pre-value has been used as covariate. The average pre-valuewas calculated from all pre-values after screening and before dosing,except for the Bristol stool scale, where the screening value was alsoused to calculate the average baseline. The average pre-value ofcitrulline was also used as covariate in the MCP-MOD, except in theSigmoid E_(max) model. This was addressed by subtracting the pre-valuetimes the slope of the linear model of the MCP part from the values ofweek 6.

Drug Dose, Drug Concentration, and Relationships to Response

The plasma apraglutide concentration against time after the first doseof apraglutide by dose level is presented in FIG. 20A-FIG. 20B. Summarypharmacokinetic parameters of apraglutide are provided in Table 26.

The mean apparent total clearance (CL/F) appeared to be constant acrossthe 3 dose levels and ranged from 16.480 (5 mg) to 20.747 L/d (10 mg)(Table 26). The mean apparent volume of distribution during the terminalelimination phase (V_(z)/F) after the last dosing appeared to be dosedependent with values of 55.426 and 105.021 L for 5 and 10 mg,respectively. Insufficient data points were available to determineV_(z)/F for the 1 mg dose group (an insufficient number of samples wereabove the lower limit of quantification during the sampling periodfollowing the last dosing).

Following the first weekly SC dose of apraglutide, plasma apraglutideconcentrations increased with no apparent lag time in all subjects(except for subject 1002 on 1 mg who had no detectable concentrations upto the next dose). The C_(max) was reached at day 1 by four, four andone subject on 1 mg, 5 mg and 10 mg apraglutide, respectively (FIG. 20).The remaining subjects reached C_(max) at day 2. Subjects on 1 mgdeclined below the limit of quantification (LOQ) at day 4 or 7 whilesubjects on 5 and 10 mg remained above the LOQ up to the next dose. Theexposure following the first dose was dose-dependent with a mean C_(max)of 13.918±11.2, 94.088±50.5 and 136.855±55.0 ng/mL for the 1, 5 and 10mg dose groups, respectively, and AUC_(tau) values of 35.214, 300.269and 476.937 d*ng/mL, respectively (Table 26). The half life (h) measuredfollowing dose #6 in the 5 mg dose group was measured as 72.7±23.0 andthe half life measured following dose #6 in the 10 mg dose group wasmeasured as 76.3±27.6.

At C_(trough) of the next 5 doses, 5 subjects (all dosed with 1 mg) hadone or multiple samples that were undetectable for apraglutide. Allother subjects showed measurable apraglutide concentrations. The meanC_(trough) over the 6 doses ranged from 0.000 (week 1) to 0.873 ng/mL(week 4) for 1 mg apraglutide, from 7.605 (week 5) to 10.163 ng/mL (week4) for 5 mg apraglutide and from 13.710 (week 6) to 18.460 ng/mL (week2) for 10 mg apraglutide (Table 26).

Following the sixth and last weekly SC dose of apraglutide, all subjects(subject 3004 was excluded because of dosing discontinuation) showedincreased apraglutide concentrations with no apparent lag time. Subjectsreached C_(max) 1 day following the last dose, except for 2 and 3subjects on 5 and 10 mg, respectively, who reached C_(max) after 2 days.Pharmacokinetic parameters were assessed up to 2 weeks following thelast dose. All subjects on 1 mg and 1 subject on 5 mg had unquantifiableconcentrations in the last 2 or 3 samples. The remaining subjects,including all subjects on 10 mg, had still quantifiable concentrationsup to 4.46 ng/mL in the last pharmacokinetic sample.

TABLE 26 Parameter 1 mg apraglutide 5 mg apraglutide 10 mg apraglutideLast dose #1-5 #6 #1-5 #6 #1-5 #6 Arithmetic mean ± standard deviationC_(max) (ng/mL) 13.918 ± 24.238 ± 94.088 ± 124.818 ± 136.855 ± 182.374 ±11.236 9.225 50.467 72.126 55.403 97.626 1^(st)/6^(th) C_(trough)(ng/ML) 0.000 ± 0.678 ± 7.912 ± 7.623 ± 14.543 ± 13.710 ± 0.000 0.7993.548 3.367 5.476 4.806 AUC_(tau) (d*ng/mL) 35.214 ± 59.437 ± 300.269 ±357.651 ± 476.937 ± 582.409 ± ±7.387 14.060 121.212 153.604 208.314284.359 AUC_(tau)/dose 0.03426 ± 0.05944 ± 0.05860 ± 0.07153 ± 0.04679 ±0.05824 ± (d*ng/mL/μg) 0.02069 0.01406 0.02505 0.03072 0.02177 0.02844C_(max)/dose (ng/mL/μg) 0.01351 ± 0.02424 ± 0.01842 ± 0.02496 ± 0.01340± 0.01824 ± 0.01087 0.00922 0.01033 0.01443 0.00580 0.00976 2^(nd)C_(trough) (ng/mL) 0.655 ± 0.742 — 9.845 ± — 18.460 ± — 4.818 8.7283^(rd) C_(trough) (ng/mL) 0.440 ± — 8.737 ± — 16.307 ± — 0.685 6.6748.960 4^(th) C_(trough) (ng/mL) 0.873 ± — 10.163 ± — 14.764 ± — 0.6836.318 8.343 5^(th) C_(trough) (ng/mL) 0.542 ± — 7.605 ± — 13.872 ± —0.867 3.944 6.968 AUC_(last) (d*ng/mL) — 56.534 ± — 382.086 ± — 623.231± 15.945 155.201 282.722 t_(1/2) (d) — *** — 3.04 ± 0.94 — 3.18 ± 1.15Lambda_Z (1/d) — *** — 0.25078 ± — 0.24632 ± 0.09871** 0.09975 CL/F(L/d) — 17.565 ± — 16.480 ± — 20.747 ± 3.818 7.341** 9.540 V_(z)/F (L) —*** — 55.426 ± — 105.021 ± 23.760** 76.365 Median (minimum, maximum)t_(max) (d) 1 0.9 1 1 1.9 1.9 (0.9, 1.9)* (0.9, 1.0) (0.9, 2.0) (0.9,2.0) (1.0, 2.0) (0.9, 2.0) Geometric mean (geometric coefficient ofvariation %) C_(max) (ng/mL) 12.314 ± 22.8 02 ± 84.086 ± 108.238 ±128.474 ± 161.910. ± 145.5 39.9 55.1 64.6 39.9 59.4* Arithmetic mean ±standard deviation 1^(st)/6^(th) C_(trough) (ng/mL) — 1.308 ± 7.232 ±6.831 ± 60.0 13.612 ± 13.024 ± 33.2 51.1 42.7 37.5* AUC_(tau) (d*ng/mL)31.555 ± 58.135 ± 279.057 ± 329.729 ± 442.982 ± 528.826 ± 145.3 23.145.0 47.2 43.3 52.4* AUC_(tau)/dose 0.03037 ± 0.05813 ± 0.05407 ±0.06595 ± 0.04291 ± 0.05288 ± (d*ng/mL/μg) 152.1 23.1 47.1 47.2 47.652.4* C_(max)/dose (ng/mL/μm) 0.01185 ± 0.02280 ± 0.01629 ± 0.02165 ±0.01245 ± 0.01619 ± 151.5 39.9 57.8 64.6 43.8 59.4* 2^(nd) C_(trough)(ng/mL) 1.289 ± — 8.960 ± — 16.775 ± — 22.11 50.4 51.6 3^(rd) C_(trough)(ng/mL) 1.315 ± 11.9 — 6.595 ± — 13.993 ± — 103.8 71.8 4^(th) C_(trough)(ng/mL) 1.306 ± 9.2 — 8.531 ± — 12.461 ± — 75.1 80.8* 5^(th) C_(trough)(ng/mL) 1.588 ± 31.2 — 6.601 ± — 11.886 ± — 69.1 79.3* AUC_(last)(d*ng/mL) — 54.708 ± — 353.984 ± — 574.308 ± 28.7 46.4 47.5* t_(1/2) (d)— *** — 2.91 ± — 3.00 ± 36.6** 41.3* Lambda_Z (1/d) — *** — 0.23840 ± —0.23103 ± 36.6** 41.3* CL/F (Lid) — 17.201 ± — 15.164 ± — 18.910 ± 23.147.2 52.4* V_(z)/F(L) — *** — 50.202 ± — 81.852 ± 61.0** 98.6*AUC_(last) = area under the plasma concentration-time curve from timezero to the last measurable concentration; AUC_(tau) = area under theplasma concentration-time curve to the end of the treatment period ofthe corresponding dosing; CL/F = apparent total clearance; C_(max) =maximum concentration; C_(trough) = plasma concentration immediatelyprior to next dosing; lambda z = terminal elimination rate constant; PK= pharmacokinetic; t_(1/2) = terminal elimination half-life; t_(max) =time to reach maximum plasma concentration; V_(z)/F = apparent volume ofdistribution during the terminal elimination phase. *1 subject missing(N = 5); **2 subjects missing (N = 4) and ***3 subjects missing (N = 3,PK parameter not calculated). Values below the limit of quantification(<1 ng/mL) were set to 0. Number of subjects is 6 unless indicatedotherwise.

The concentration profile observed following the sixth dose appeared tobe comparable to the first dose, although the exposure was slightlyhigher (FIG. 20). Exposure indicated dose proportionality with a meanC_(max)/dose of 0.02424, 0.02496 and 0.01824 ng/mL/μg and AUC_(tau)/doseof 0.05944, 0.07153 and 0.05824 d*ng/mL/μg, for the 1, 5 and 10 mg dosegroups, respectively (Table 26).

In 3 out of 6 subjects dosed with 1 mg, insufficient data points wereavailable to determine the terminal elimination rate constant (aninsufficient number of samples were above the lower limit ofquantification during the sampling period following the last dosing).Subsequently, the mean half-life was determined only for the 5 and 10 mgdose groups and was comparable between the two groups (3.04 and 3.18days, respectively).

The variability in C_(max) and AUC_(tau) was noticeable following thefirst dose of 1 mg with a % CV of 145.5 and 145.3%, respectively (Table26). Following the last dose of 1 mg and both the first and last dose ofthe 5 and 10 mg dose levels the variability was lower; the % CV forC_(max) ranged from 39.9 to 64.6% and the corresponding range for % CVfor AUC_(tau) was 23.1 to 52.4%.

Visual inspection of the dose-normalized pharmacokinetic parametersC_(max) and AUC_(tau) values suggested that over the 1 to 10 mg doserange, apraglutide follows linear kinetics. This is in line with theaccompanying constant CL/F.

As with citrulline, a statistical steady-state analysis per dose levelwas performed for apraglutide using Helmert's approach. Thissteady-state analysis revealed that none of the contrasts werestatistically significant, except for the mean C_(trough) over week 3 to6 for 10 mg against mean C_(trough) at week 2 (−3.616, 95% CI: −5.893;−1.399 and p=0.0023). Visual inspection of the data suggests that steadystate was reached following the first dosing at all investigated doselevels.

Graphical analysis was performed to assess the apraglutideconcentration-citrulline effect relationship. Several individualcitrulline parameters (R_(max) and R_(pre-dose)) were plotted againstapraglutide pharmacokinetic parameters (C_(max), C_(trough) andAUC_(tau)). For the parameters assessed at dose 6, a regression line wasgenerated. The p-value of the slope almost reached significance forC_(max) against R_(max) with a value of 0.0608 (FIG. 21). AUC_(tau)appeared to be positively correlated with R_(max) with a p-value of0.0377 (FIG. 22). According to the regression line, for each 100 d*ng/mLincrease in AUC_(tau) in week 6, the corresponding maximum citrullineresponse increased by 0.39 μg/mL. The R squared value showed that 26% ofthe variability in R_(max) was explained by AUC_(tau). As shown in FIGS.23, 24, and 25, the correlation plots of all apraglutide and citrullineconcentrations assessed in week 6 by dose level revealedcounter-clockwise hysteresis as there was a time delay between themeasured apraglutide concentration and the citrulline effect.

Pharmacokinetics

The mean CL/F was constant across the 3 dose levels and ranged from16.480 (5 mg) to 20.747 L/d (10 mg). The mean Vz/F was determined onlyfor the 5 and 10 mg dose groups and was dose dependent with values of55.426 and 105.021 L for 5 and 10 mg, respectively. The mean t½ could bedetermined only for the 5 and 10 mg apraglutide dose groups and wascomparable for these groups (3.04 and 3.18 days, respectively).

The exposure following the first weekly SC dose of apraglutide indicateddose proportionality with a mean C_(max) of 13.918, 94.088 and 136.855ng/mL for the 1, 5 and 10 mg dose groups, respectively, and AUC_(tau)values of 35.214, 300.269 and 476.937 d*ng/mL, respectively. Theconcentration profile observed following the sixth dose was comparableto the first dose, although the exposure was numerically higher.Exposure indicated dose proportionality with a mean C_(max)/dose of0.02424, 0.02496 and 0.01824 ng/mL/μg and AUC_(tau)/dose of 0.05944,0.07153 and 0.05824 d*ng/mL/μg, for the 1, 5 and 10 mg dose groups,respectively. At dose 1 and 6, t_(max) was approximately 1 day for the 1mg and 5 mg apraglutide doses and 2 days for the 10 mg dose.

Visual inspection of the dose-normalized pharmacokinetic parametersC_(max) and AUC_(tau) values suggests that over the 1 to 10 mg doserange, apraglutide follows linear kinetics. Visual inspection of thedata suggests that steady state was reached following the first dosingat all investigated dose levels.

Pharmacodynamics

Following the first and last dosing with apraglutide, citrullineconcentrations dose-dependently increased. The mean citrulline increaseobserved following the last dose appeared to decline more rapidly forthe 1 mg and 5 mg doses compared to the 10 mg dose. Rt1/2 could only beassessed for 2 subjects, both dosed with 10 mg, and was found to be 6.5and 11.0 days.

At dose 6, the mean R_(max) was 7.1702, 8.1577 and 8.7254 μg/mLfollowing the last dose of 1, 5 and 10 mg, respectively, and was reachedat 2.0, 3.9 and 3.9 days. The corresponding placebo value was 6.3707μg/mL at 16.9 days following the last dose. These R_(max) values werecomparable with the mean R_(max) over the whole trial period. Visualinspection of the data suggests that steady state was reached followingthe first dosing at all investigated dose levels.

A significant treatment effect of apraglutide was observed forcitrulline (p=0.0007). Apraglutide induced an increase in citrulline.This response was statistically significant with an increase of 1.2574μg/mL for 5 mg apraglutide (95% CI: 0.5037; 2.0112; p=0.0025) and 1.6343μg/mL for 10 mg apraglutide (95% CI: 0.8809; 2.3878; p=0.0002) comparedto placebo; for 1 mg apraglutide the increase compared to placebo wasnot statistically significant (0.3146 μg/mL, 95% CI: −0.4371; 1.0663;p=0.3910)). The contrast between dose groups indicated that the 5 and 10mg doses induced significantly higher citrulline levels compared to the1 mg dose (0.9429 μg/mL, 95% CI: 0.1768; 1.7089; p=0.0186 and 1.3198μg/mL, 95% CI: 0.5586; 2.0810; p=0.0018, respectively). Importantly, thedifference between 5 and 10 mg apraglutide was not statisticallysignificant, although the effect following 10 mg appeared to be slightlyhigher than following 5 mg.

A significant treatment effect of apraglutide on the Bristol stool scalewas observed with a p-value of 0.0189 (all dose groups compared toplacebo). This effect was not dose-dependent as only a significantdecrease compared to placebo was observed for the 1 and 5 mg dose groups(−0.9, 95% CI: −1.6; −0.3; p=0.0092, and −1.0, 95% CI −1.7; −0.4;p=0.0046, respectively).

Exploratory graphical analysis revealed that the slope of the regressionline for C_(max) against R_(max) at dose 6 almost reached significancewith a p-value of 0.0608. AUC_(tau) appeared to be positively correlatedwith R_(max) with a p-value of 0.0377. According to the regression line,for each 100 d*ng/mL increase in AUC_(tau) in week 6, the correspondingmaximum citrulline response increased by 0.39 μg/mL. The R squared valueshowed that 26% of the variability in R_(max) was explained byAUC_(tau). The correlation plot of all apraglutide and citrullineconcentrations assessed in week 6 revealed counter-clockwise hysteresis.

The MCP-MOD procedure showed that 3 candidate pre-specified modelsyielded similar results for best fitting the dose response in the MCPpart; the 1) E_(max), 2) Log Lin and 3) Sigmoid E_(max) model. These 3models were all applied in the MOD part and predicted similar citrullinechanges from placebo for a dose of 1, 5 and 10 mg apraglutide. With adose of 1 mg, the predicted placebo and baseline corrected change incitrulline across the 3 models ranged from 0.7125 to 0.8712 μg/mL withsignificant results for the E_(max) (p=0.0184) and Log Lin (p=0.0238)models. For the Sigmoid E_(max) model, the p-value was 0.0645. For adose of 5 mg, the response ranged from 2.1652 to 2.3327 μg/mL. Aslightly stronger response was obtained for the 10 mg dose, ranging from2.6946 to 2.8604 μg/mL. The predicted response versus placebo for both 5and 10 mg was statistically significant for all models, with a p-valueof <0.0001.

The apraglutide dose levels required for a baseline and placebocorrected citrulline increase of 1, 2 and 3 μg/mL were predicted. TheE_(max) of the Sigmoid E_(max) model was <3 μg/mL, so for this model achange of 2.5 μg/mL was set as the largest response. The predictedapraglutide dose for a citrulline increase of 1 μg/mL was 1.2333 mg (95%CI: −0.04246; 2.5091), 1.2249 mg (95% CI: −0.2169; 2.6667) and 1.3606 mg(95% CI: 0.2680; 2.4532), for the E_(max), Log Lin and Sigmoid E_(max)model, respectively. To evoke a citrulline increase of 2 μg/mL, thepredicted apraglutide doses were 3.8471 mg (95% CI: 0.3534; 7.3409),4.2011 mg (95% CI: 0.6406; 7.7617) and 3.4042 mg (95% CI: −0.1290;6.9374) for the E_(max), Log Lin and Sigmoid E_(max) model,respectively. The predicted apraglutide doses for change from placebo of2.5 or 3 μg/mL were 13.1053 mg (95% CI: −6.3068; 32.5173), 11.4325 mg(95% CI: 1.5808; 21.2843) and 6.4619 mg (95% CI: −0.5521; 13.4759) usingthe E_(max), Log Lin and Sigmoid E_(max) model, respectively.

PD/PK Model

Clinical pharmacokinetic observations were best matched by a model withcorrelation between V1/F and Cl/F and a dose covariate on absorptionduration as well as body weight covariates on V1/F and Cl/F.Pharmacodynamic observations were then added to this model to create thePK/PD model. Plasma citrulline was described by a turnover model andmaximal PD effect model.

The population PK/PD model estimated that a 70-kg individual whoreceived apraglutide 5 mg subcutaneous injection would achieve a volumeof distribution of 31.3 L and peak plasma concentration (C_(max)) at1.39 days. Simulated apraglutide and citrulline plasma concentrationprofiles with weekly subcutaneous injection of apraglutide 2.5, 5, or 10mg are shown in FIG. 26.

The PK/PD model did not indicate any accumulation of apraglutide overtime. Although accumulation of citrulline was apparent during the firstthree weeks of treatment, a steady state concentration was subsequentlyreached. Simulated apraglutide concentration-time profiles by bodyweight indicated a lower area-under-the curve (AUC) and C_(max) atsteady state with increasing body weight.

Example 8: Apraglutide Manufacturing Process—(II)

The following is an exemplary method of the present disclosure for themanufacture of apraglutide at improved levels of purity relative topreviously described synthesis routes (e.g. U.S. Pat. No. 8,580,918).

Solid Phase Peptide Synthesis (Step 1)

SPPS is the sequential synthesis of a peptide chain anchored on a solidsupport by repetition of a cycle encompassing the following steps:

-   -   1. Removal of the N-terminus Fmoc protecting group of the        peptide resin        -   1a. In some aspects, the Fmoc deprotection reaction of            residue Asp³ comprises treating the resin with a solution of            10% piperidine and 2% Oxyma in DMF. The deprotection            reaction is performed in two cycles, the first one for 15            minutes and the second one for 30 minutes.    -   2. DMF washes    -   3. Couplings of Fmoc-AA-OH    -   4. Coupling test    -   5. DMF washes

This cycle is repeated until the peptide sequence is completed.

The α-amino groups of the amino acids are protected with thebase-sensitive 9-fluorenylmethyloxycarbonyl (Fmoc) group; the side chainfunctional groups are protected with acid-labile groups. All amino acidsderivatives used in the process are commercially available.

SPPS is the sequential synthesis of a peptide chain anchored on a solidsupport. In the synthesis, MBHA resin may be used to assemble thepeptide sequence. After swelling and washing the resin with DMF and thenwith DMF/DIEA under nitrogen atmosphere the Fmoc-Rink-amide linker maybe coupled using HBTU/DIPEA/HOBt in DMF. After coupling, the resin maybe washed with DMF and then acetylated using Ac₂O/DIPEA. A Kaiser testmay be carried out to check completion of the coupling.

After washing the resin with DMF, the Fmoc protected amino acids areeach coupled to the resin-bond peptide according to the following cycle:

1. The Fmoc-protecting group is removed with piperidine in DMF and theresin is washed thoroughly with DMF.2. The coupling is performed in DMF with variable amino acid equivalentsusing DIC/oxyma for activation.

-   -   2a. In some aspects, the di-peptide Boc-His(Trt)-Gly-OH is        coupled to the resin instead of sequential assembly of Gly and        His amino acids. The dipeptide is preactivated for 1 hour at 20°        C.±2° C. using Boc-His (Trt)-Gly-OH/Oxyma/DIC (2.5 mmol/2.5        mmol/2.5 mmol) in 7 mL DMF, before addition to the coupling        reaction. A Kaiser test may be carried out to check completion        of the coupling.        3. Coupling of amino acids is monitored by using the Ninhydrin        assay, which is performed during each synthesis cycle.

At the end of the assembly, after the last amino acid has been coupledand deprotected, the resin is washed with DMF and isopropanol, and driedunder vacuum.

Cleavage of the Peptide from the Resin and Deprotection (Step 2)

The protected peptide may be simultaneously cleaved from the resin anddeprotected by treatment with a mixture TFA/water/anisole. MTBE issubsequently added to the peptide/TFA slurry to precipitate the crudepeptide in the presence of cleaved resin. The obtained crude peptide isfiltered, washed with MTBE and dried under vacuum to constant weight.

Decarboxylation Reaction (Step 3a)

The crude peptide is solubilized in a mixture of H₂O/ACN (70:30 ratio)in ammonia buffer. The solution is adjusted to target pH 8.0±0.1 using25% acetic acid or 25% NH₄OH in H₂O. The decarboxylation reaction ismaintained at 50° C. for 65 minutes. The crude peptide is washed with asolution of H₂O/ACN (70:30 ratio) in ammonia buffer at pH 8.0±0.1 andstored at 5°±3° C.

Purification by Preparative RP-HPLC (Step 3b)

The crude peptide is dissolved in a mixture of water/acetonitrile/NH₄OH.This solution is diluted with acetic acid and then filtered.

The primary purification is conducted on preparative RP-HPLC withNaHCO₃/H₂O/CH₃CN as eluent. The elution from the column is monitored byUV and the fractions obtained are analyzed by RP-HPLC. Fractions meetingthe monitoring criteria are mixed in the combined pool. Fractions notmeeting the monitoring criteria may be recycled by repeating thepurification step. The purity of the pool is controlled by analyticalRP-HPLC.

Sodium Salt Conversion by Preparative RP-HPLC (Step 4)

This step may be conducted to exchange the counter ion of the peptidefrom a TFA anion to a sodium cation through a pH change and to furtherpurify the peptide. The combined pool from Step 3 is diluted in waterand re-purified by preparative RP-HPLC using NaOAc eluent.

The purified peptide solution is subsequently subject to evaporationunder vacuum to reduce acetonitrile in the solution. The purifiedpeptide solution is then adjusted to target pH 7.9 using 0.1% AcOH.

The pure pool may be concentrated and freeze-dried. The purity of thepool is analyzed by RP-HPLC.

Freeze-Drying and Packaging (Step 5)

Prior to lyophilization, the purified peptide in solution is filteredthrough a 0.2 μm membrane. The lyophilization is carried out at lowpressure. The resulting lyophilized final peptide is packed under argon.The lyophilized apraglutide is controlled according to the apraglutidespecification.

Reprocessing

Lyophilized apraglutide that does not fulfil the criteria established inthe apraglutide specification may be subjected to re-purification.

Re-purification may be carried out after reconstitution of the peptideby repeating the purification step(s) and counterion conversion step, asdescribed above.

After re-purification, the material is lyophilized according to theprocedure described above.

Lyophilized apraglutide that does not fulfil the criteria established inthe apraglutide specification may be subjected to re-lyophilization.

Re-lyophilization may be carried out after reconstitution of the peptideby repeating the lyophilization step, as described above.

Impurities

The apraglutide manufacturing process as described in Example 8 wasperformed to reduce the level of β-Asp³ peptide isomer impurity inapraglutide below 1.5%. In some embodiments, the apraglutidemanufacturing process as described in Example 8 reduced the level ofβ-Asp³ peptide isomer impurity in apraglutide below 1%.

Formation of the β-Asp³ impurity is favored by high pH and the presenceof the sequence Asp³-Gly⁴. The Asp-Gly sequence is particularly prone toaspartimide formation, resulting from a ring-closure (attack of thenitrogen from the α-carboxy amide bond on the β-carboxy side chain).Aspartimides are susceptible to base-catalyzed epimerization and mayundergo ring-opening reactions, which may lead to the formation ofmultiple by-products. Attack by water may produce the β-aspartylpeptide.

The formation of β-Asp³ impurity during the decarboxylation/extractionstep (Step 3a) upon peptide cleavage from resin can reach levels of >1%and is promoted by prolonged exposure to extreme pHs, in particular,strong base solution of ammonia in H₂O/ACN 80:20, pH around 10 at roomtemperature. The apraglutide manufacturing process as described inExample 8 reduced the pH from 10 to 8, shortened the decarboxylationreaction time from 24 hours to 65 minutes, changed the reactiontemperature from Room Temperature to 50° C., and reduced the storagetemperature from Room Temperature to 5° C. These changes resulted inβ-Asp³ impurity at levels below 1.5%. In some embodiments, these changesresulted in β-Asp³ impurity at levels below 1%.

The apraglutide manufacturing process as described in Example 8 amendedthe Fmoc deprotection reaction of residue Asp³ to use a solution of 10%piperidine and 2% Oxyma in DMF with two cycles of deprotection, thefirst one for 15 minutes and the second one for 30 minutes. These milderbasic conditions allowed a complete deprotection of Asp³ and decreasethe aspartimide and subsequent β-Asp³ by-product formation.

The formation of a D-His impurity may occur due to the racemization ofthe amino acid during the coupling reaction using DIC/Oxyma. A processof sequential assembly of individual Gly and His amino acids showedvariability in levels of D-His impurity between 0.3%-1.2%, sometimeswith incomplete coupling. Fmoc-His(Trt)-OH incorporation using thecoupling reagents DIC/Oxyma may favor the racemization into the D formof His.

The apraglutide manufacturing process as described in Example 8 used thedi-peptide Boc-His(Trt)-Gly-OH as starting material for this reactioninstead of sequential assembly of Gly and His amino acids. This allowsdecreasing the racemization level in His to below 1% (0.3% in oneperformed batch) and incorporates the final D-His level to thespecifications of the dipeptide (Boc-D-His(Trt)-Gly-OH). This alsoallows a reduction in the number of deprotection steps, thus minimizingthe contributing impact of deprotection conditions (high pH) to theβ-Asp³ impurity formation, as described above.

Example 9: Phase III Human Clinical Trial of Administration ofApraglutide Compositions of the Present Disclosure

The following non-limiting example describes a Phase 3 clinical trial inwhich subjects with SBS-IF are treated with the apraglutide formulationsof the present disclosure. This Phase 2 clinical trial investigatesefficacy of weekly subcutaneous apraglutide in reducing parenteralsupport dependency in patients with SBS-IF.

144 adult patients with SBS-IF will be treated with apraglutidecompositions of the present disclosure of placebo. Patients with SBS-IFwill be administered 2.5 mg apraglutide of the present disclosure whenthe subject has a body weight of less than 50 kg, or 5 mg apraglutide ofthe present disclosure when the subject has a body weight greater thanor equal to 50 kg, or placebo once a week for 48 weeks.

The trial is a multicenter, double-blind, randomized,placebo-controlled, phase 3 trial. Randomization of participantsincludes stoma and colon-in-continuity anatomy-specific randomization.

Primary endpoint efficacy assessments will be performed at 24 weeks. Theprimary endpoint will evaluate the relative change from baseline inactual weekly PS volume. Secondary endpoint efficacy assessments will beperformed at 24 weeks and 48 weeks. Anatomy specific secondary endpointsas well as secondary endpoints common across all patients will beevaluated.

The secondary endpoints to be evaluated, include, but are not limitedto:

1. Subjects who achieve a reduction of at least 1 day per week of PS atWeeks 24/48.2. Relative change from baseline in actual weekly PS volume at Weeks12/24/48.3. SBS-IF patients reaching enteral autonomy at Weeks 24/48.4. At least 20% reduction of PS volume from baseline at Weeks 20/24.5. Calorie reduction in the parenteral nutrition (PN) at Weeks 24.6. Change from baseline on the Patient Global Impression of Severity(PGIS)7. Change from baseline on the Pittsburgh Sleep Quality Inventory (PSQI)8. Change from baseline on the Patient Global Impression of Change(PGIC)9. Absorption rate constant (ka) of apraglutide through population PKdata analysis10. Apparent clearance (CL/F) of apraglutide through population PK dataanalysis11. Apparent volume of distribution (Vz/F) of apraglutide throughpopulation PK data analysis

Example 10: Phase I Human Clinical Trial of Administration ofApraglutide Compositions of the Present Disclosure

The following non-limiting example describes a Phase I clinical trial inwhich subjects with normal and impaired kidney function are treated withthe apraglutide formulations of the present disclosure. This Phase Iclinical trial investigates the pharmacokinetics and safety of a singlesubcutaneous dose of 5 mg apraglutide in subjects with varying degreesof renal function. The renal function is calculated by the estimatedglomerular filtration rate (eGFR) according to the Chronic KidneyDisease Epidemiology (CKD-EPI) Creatinine Equation.

The trial is a two-stage, open label, multi-center, non-randomized,Phase I trial. In Part 1 of the trial, 8 subjects with severe renalimpairment (Cohort 1) and 6 subjects with normal renal function (Cohort2) will be administered a single dose 5 mg apraglutide of the presentdisclosure. In Part 2 of the trial, 8 subjects with moderate renalimpairment (Cohort 3) and 8 subjects with mild renal impairment (Cohort4) will be administered a single dose 5 mg apraglutide of the presentdisclosure. Subjects will be enrolled if the geometric mean ratio (GMR)of AUC_(inf) or AUC_(last) for the severe renal impairment groupcompared to the control group is ≥2.

Patient eligibility is assessed during a screening visit. The maininclusion criteria are:

1. All Participants

-   -   a. Age between 18 and 75 years inclusive    -   b. Subjects who are willing and able to comply with the study        procedures    -   c. Subjects able to understand and willing to sign the informed        consent    -   d. Body mass index (BMI) of ≥17.5 to ≤40 kg/m2; and a total body        weight of >50 kg (110 lb).    -   e. Women of childbearing potential (WOCBP) on highly effective        method of contraception during the trial and for 1 month after        the end of trial (EOT) visit. Sterilized or infertile or        postmenopausal females.    -   f. Male subjects with a female partner of childbearing        potential: highly effective methods of contraception and no        sperm donation during the trial and for 1 month after (EOT)        visit.        2. Healthy participants    -   a. No clinically relevant abnormalities (medical history, vital        signs, ECG, safety labs)    -   b. eGFR measured by CKD-EPI≥90 mL/min/1.73 m2 at two screening        visits    -   c. Demographically comparable to the group of subjects with        impaired renal function        3. Participants with impaired renal function    -   a. Severe renal impairment: eGFR<30 mL/min/1.73 m2, but not        requiring hemodialysis    -   b. Moderate renal impairment: eGFR≥30 mL/min/1.73 m2 and <60        mL/min/1.73 m2    -   c. Mild renal impairment: eGFR≥60 and <90 mL/min/1.73 m2

Primary endpoint efficacy assessments will be performed at 240 hoursafter single dose. The primary endpoint will evaluate maximum plasma(C_(max)), area under the concentration-time curve from 0 to infinity(AUC_(inf)) and area under the concentration-time curve up to lastmeasurable concentration (AUC_(last)), from time 0 to 240 hours.Secondary endpoint efficacy assessments will be performed at 240 hours,7 days, 14 days, and 28 days. Primary and secondary endpoints will beevaluated across all subjects.

The secondary endpoints to be evaluated, include, but are not limitedto:

-   1. Area under the concentration-time curve up to the last measurable    concentration from time 0 to 7 days (AUC_(0-7days)). Samples    collected over 168 hours after single dose-   2. Time to maximum concentration (T_(max)). Samples collected over    240 hours after single dose-   3. Terminal elimination rate constant. Samples collected over 240    hours after single dose-   4. Terminal elimination half-life. Samples collected over 240 hours    after single dose-   5. Apparent clearance after extravascular administration (CL/F).    Samples collected over 240 hours after single dose-   6. Apparent volume of distribution after extravascular    administration (Vz/F). Samples collected over 240 hours after single    dose-   7. Number of participants with adverse events or adverse events of    special interest at days 14-28-   8. Clinically significant change from baseline in vital signs at    days 14-28-   9. Clinically significant change from baseline in recorded    triplicate 12-lead ECG at days 14-28-   10. Number of participants who experience a clinically significant    change from baseline in clinical laboratory assessments at days    14-28

As would be appreciated by the skilled artisan, patients with renalimpairment usually require drug dosing adjustments because renalimpairment can adversely affect some pathways of hepatic/gut drugmetabolism, and has also been associated with other changes such aschanges in absorption, plasma protein binding, transport, and tissuedistribution. Even if pharmacokinetic data indicated that renalexcretion is not the primary route of elimination for apraglutide, otherGLP-2 analogues (i.e. teduglutide) necessitate dose reduction inpatients with moderate and severe renal impairment and end-stage renaldisease. In the trial described in Example 10, it has been found that itis safe to dose the patients with severe CKD with apraglutide 5 mg,without the risk of overdose in this population. Without wishing to bebound by theory, this indicates that apraglutide has the potential ofbeing administered to renal impaired patients without dose adaptation.

Pharmacokinetics

The plasma concentration of apraglutide after administration of a singlesubcutaneous dose of 5 mg apraglutide was monitored over a period of 240hours in both healthy subjects (Cohort 2) and subjects with severe CKD(Cohort 1) and is presented in FIG. 29.

Summary of pharmacokinetic parameters of apraglutide are provided inTable 27. Two outliers were identified (one in each Cohort) whenanalyzing the individual apraglutide plasma concentration curves. Theoutlier in Cohort 1 had the highest bodyweight (BW) and body mass index(BMI) in the group while the outlier in Cohort 2 had the lowest BW andBMI in the group. The geometric mean rations were calculated with andwithout the outlier subjects.

TABLE 27 Single dose 5 mg apraglutide 90% CI Geometric 90% CI ParameterLower Bound Mean Ratio Higher Bound Whole Cohort C_(max) (ng/mL) 0.3700.572 0.885 AUC_(inf) (h*ng/mL) 0.388 0.614 0.972 Cohort without the twooutliers C_(max) (ng/mL) 0.556 0.774 1.077 AUC_(inf) (h*ng/mL) 0.6570.849 1.098 C_(max) = maximum plasma; AUC_(inf) = area under theconcentration-time curve from 0 to infinity

For the entire subject population, the geometric mean ratio for C_(max)was 0.572 with 90% CI 0.37-0.885 and for AUC_(inf) was 0.614 with 90% CI0.388-0.972. Whereas, when removing the data from the outlier subjectsthe geometric mean ratio for C_(max) was 0.774 with 90% CI 0.556-1.077and for AUC_(inf) was 0.849 with 90% CI 0.657-1.098. As would beappreciated by the skilled artisan, patients with severe renalimpairment have higher bodyweight and body mass index than healthysubjects. Without wishing to be bound by theory, this may indicate thatthe subjects in Cohort 1 have lower exposure to apraglutide due to lowerplasma concentrations.

Example 11: Apraglutide Manufacturing Process—(III)

FIG. 2A and FIG. 2B schematically depict an exemplary method of thepresent disclosure for the manufacture of apraglutide at improved levelsof purity relative to previously described synthesis routes (e.g. U.S.Pat. No. 8,580,918). The manufacture process described in FIG. 2A andFIG. 2B is herein referred to as Process B and incorporates primarypurification by RP-HPLC (C18) chromatography in TFA-based mobile phases(H₂O/acetonitrile) to ≥90% purity with pH of fractions adjusted usingsodium bicarbonate (NaHCO₃), followed by secondary purification byRP-HPLC (C18) in NaHCO₃ mobile phases (H₂O/acetonitrile) to ≥97% purity,and followed by desalting/buffer exchange by RP-HPLC (C18) in sodiumacetate (NaOAc)/H₂O/acetonitrile mobile phases. Table 2a shows thepurity of Apraglutide product produced using Process B, as well as thelevel of major contaminants. Table 2a shows the purity of Apraglutideproduct produced using Process B, as well as overall product yields forthe process. As shown in Table 2a and Table 2b, Process B can yieldApraglutide that has a purity of no less than 97%, and low levels ofcontaminants. Moreover, Process B exhibits product yields of about 20%.

Impurities

The apraglutide manufacturing process as described in FIG. 2A and FIG.2B can be further modified to reduce the level of β-Asp³ peptide isomerimpurity in apraglutide below 1.5%. In some embodiments, the apraglutidemanufacturing process as described in Example 8 reduced the level ofβ-Asp³ peptide isomer impurity in apraglutide below 1%.

Formation of the β-Asp³ impurity is favored by high pH and the presenceof the sequence Asp³-Gly⁴. The Asp-Gly sequence is particularly prone toaspartimide formation, resulting from a ring-closure (attack of thenitrogen from the α-carboxy amide bond on the β-carboxy side chain).Aspartimides are susceptible to base-catalyzed epimerization and mayundergo ring-opening reactions, which may lead to the formation ofmultiple by-products. Attack by water may produce the β-aspartylpeptide.

The formation of β-Asp³ impurity during the decarboxylation/extractionstep (Step 3(1), FIG. 2B) upon peptide cleavage from resin can reachlevels of >1% and is promoted by prolonged exposure to extreme pHs, inparticular, strong base solution of ammonia in H₂O/ACN 80:20, pH around10 at room temperature. The apraglutide manufacturing process asdescribed in FIG. 2A and FIG. 2B can be modified as to reduce the pHfrom 10 to 8, shorten the decarboxylation reaction time from 24 hours to65 minutes, change the reaction temperature from room temperature to 50°C., and reduce the storage temperature from room temperature to 5° C.These changes can result in β-Asp³ impurity at levels below 1.5%. Insome embodiments, these changes resulted in β-Asp³ impurity at levelsbelow 1%.

The apraglutide manufacturing process as described in FIG. 2A and FIG.2B can also be modified such that the Fmoc deprotection reaction ofresidue Asp³ to use a solution of 10% piperidine and 2% Oxyma in DMFwith two cycles of deprotection, the first one for 15 minutes and thesecond one for 30 minutes. These milder basic conditions allow acomplete deprotection of Asp³ and decrease the aspartimide andsubsequent β-Asp³ by-product formation.

The formation of a D-His impurity may occur due to the racemization ofthe amino acid during the coupling reaction using DIC/Oxyma. A processof sequential assembly of individual Gly and His amino acids showedvariability in levels of D-His impurity between 0.3%-1.2%, sometimeswith incomplete coupling. Fmoc-His(Trt)-OH incorporation using thecoupling reagents DIC/Oxyma may favor the racemization into the D formof His.

The apraglutide manufacturing process as described in FIG. 2A and FIG.2B can be modified to use the di-peptide Boc-His(Trt)-Gly-OH as startingmaterial for this reaction instead of sequential assembly of Gly and Hisamino acids. This allows decreasing the racemization level in His tobelow 1% (0.3% in one performed batch) and incorporates the final D-Hislevel to the specifications of the dipeptide (Boc-D-His(Trt)-Gly-OH).This also allows a reduction in the number of deprotection steps, thusminimizing the contributing impact of deprotection conditions (high pH)to the β-Asp³ impurity formation, as described above.

The apraglutide manufacturing process as described in FIG. 2A and FIG.2B, and modified as described above, is herein referred to as “ProcessC”. As shown in Table 2b, Process C can yield Apraglutide that has apurity of no less than 97% and exhibits product yields of about 22%.

EQUIVALENTS

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the disclosure to the preciseform disclosed. The details of one or more embodiments and/or aspects ofthe disclosure are set forth in the accompanying description above. Anyone of the embodiments and/or aspects described herein can be combinedwith any other embodiment and/or aspect described herein, and any numberof embodiments and/or aspects can be combined. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferred methodsand materials are now described. Other features, objects, and advantagesof the disclosure will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural referents unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. All patents and publicationscited in this specification are incorporated by reference.

What is claimed is:
 1. A sodium salt of apraglutide having a purity ofno less than 95%, wherein apraglutide has the following structure:


2. The sodium salt of apraglutide of claim 1, wherein the sodium salt ofapraglutide has a purity of no less than 97%.
 3. The sodium salt ofapraglutide of any one of the preceding claims, wherein the sodium saltof apraglutide comprises no more than 3% of a Des-Gly⁴ apraglutideimpurity.
 4. The sodium salt of apraglutide of any one of the precedingclaims, wherein the sum of Aspartimide³ apraglutide, Asp³³-OHapraglutide and Des-Ser⁷ apraglutide impurities in the sodium salt ofapraglutide is no more than 2%.
 5. The sodium salt of apraglutide of anyone of the preceding claims, wherein the sodium salt of apraglutidecomprises no more than 2% of a [Trp²⁵, 2-(2,4,6-trimethoxyphenyl)]apraglutide impurity.
 6. The sodium salt of apraglutide of any one ofthe preceding claims, wherein the sodium salt of apraglutide comprisesno more than 1.5% of a β-Asp³ apraglutide impurity.
 7. The sodium saltof apraglutide of any one of the preceding claims, wherein the sodiumsalt of apraglutide comprises no more than 1% of a β-Asp³ apraglutideimpurity.
 8. The sodium salt of apraglutide of any one of the precedingclaims, wherein the sodium salt of apraglutide comprises no more than 1%of a D-His apraglutide impurity.
 9. The sodium salt of apraglutide ofany one of the preceding claims, wherein the sodium salt of apraglutidecomprises: no more than 1% of a Asp³³-OH apraglutide impurity, no morethan 1% of a Des-Ser⁷ apraglutide impurity, no more than 1% of aD-Aspartimide³ apraglutide impurity, no more than 1% of a [Trp²⁵,2-(2,4,6-trimethoxyphenyl)] apraglutide impurity, and wherein the sum ofDes-Gly⁴ apraglutide and Aspartimide³ apraglutide impurities in thesodium salt of apraglutide is no more than 1%.
 10. The sodium salt ofapraglutide of any one of the preceding claims, wherein the sodium saltof apraglutide is provided as a lyophilized powder.
 11. A pharmaceuticalcomposition comprising the sodium salt of apraglutide of any one of thepreceding claims.
 12. The pharmaceutical composition of claim 11,further comprising at least one of glycine, L-histidine and mannitol.13. A pharmaceutical composition of claim 12, wherein the pharmaceuticalcomposition comprises: about 12.5 mg of apraglutide (sodium salt); about1.88 mg of glycine; about 3.88 mg of L-histidine; about 57.5 mg ofmannitol.
 14. The pharmaceutical composition of claim 12 or claim 13,wherein the pharmaceutical composition is provided as a lyophilizedpowder.
 15. A two-chamber powder syringe comprising the sodium salt ofapraglutide of any one of claims 1-10 or the pharmaceutical compositionof any one of claims 11-14.
 16. A method of making a GLP-2 analogpeptide comprising: a) performing solid phase peptide synthesis (SPPS)to synthesize the GLP-2 analog peptide on anFmoc-Rink-amid-MethylBenzHydril Amine(MBHA)-resin; b) cleaving thesynthesized GLP-2 analog peptide off the resin and deprotecting the sidechains of the synthesized GLP-2 analog peptide by treating the resinwith a solution comprising trifluoroacetic acid (TFA), water, andanisole; c) purifying the synthesized GLP-2 analog peptide from step (b)by performing a first preparative reversed-phase high performance liquidchromatography (RP-HPLC) purification using TFA-based mobile phases,thereby producing a solution comprising the GLP-2 analog peptide with apurity of no less than 90%; d) purifying the product of step (c) byperforming a second RP-HPLC purification, using NaHCO₃-based mobilephases, thereby producing solution comprising the GLP-2 analog peptidewith a purity of no less than 97%.
 17. The method of claim 16, furthercomprising: e) further purifying the product from step (d) by performinga third RP-HPLC purification using NaOAc-based mobile phases, therebyproducing a solution comprising the sodium salt of the GLP-2 analogpeptide with a purity of no less than 97%.
 18. The method of claim 17,further comprising: f) adjusting the pH solution comprising the sodiumsalt of the GLP-2 analog peptide to about pH 7.9 using 0.1% AcOH inwater; g) passing the product of step (f) through a filter with a poresize of 0.2 μm; h) lyophilizing the product of step (g), therebyproducing lyophilized sodium salt of the GLP-2 analog peptide with apurity of no less than 97%.
 19. The method of claim 16, furthercomprising: c)(i) performing a decarboxylation of the synthesized GLP-2analog peptide by solubilizing the peptide in a solution comprisingwater and acetonitrile in ammonia buffer.
 20. The method of claim 19,wherein the pH of the solution comprising water and acetonitrile inammonia buffer is adjusted to about pH 8.0.
 21. The method of any one ofclaims 16-20, wherein step (a) comprises: i) preparing a MBHA-resin onwhich the SPPS will be performed ii) performing an initial Fmocdeprotection reaction followed by a coupling reaction to add a firstFmoc-protected amino acid to the resin, thereby forming a protectedpeptide on the resin; iii) performing an Fmoc deprotection reactionfollowed by a coupling reaction to append at least one Fmoc-protectedamino acid to the protected peptide; iv) repeating step iii until theGLP-2 analog peptide is synthesized on the resin to produce aFmoc-protected and side-chain protected GLP-2 analog peptide linked tothe resin; v) performing an Fmoc deprotection reaction to produce aside-chain protected GLP-2 analog peptide linked to the resin; and vi)drying the side-chain protected GLP-2 analog peptide linked to theresin.
 22. The method of claim 21, wherein step (a)(i) comprises: (a1)washing the resin with a solution comprising dimethylformamide (DMF) andN,N-Diisopropylethylamine (DIEA) at 5 mL of solution per gram of resinunder an N₂ atmosphere; (b1) coupling a Rink amide linker to the resinin a solution comprising2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,Hexafluorophosphate Benzotriazole Tetramethyl Uronium (HBTU), DIEA andHydroxybenzotriazole (HOBt) in DMF; (c1) washing the product formed instep (b1) with DMF (d1) performing a reduction reaction by contactingthe resin with a solution comprising acetic anhydride (Ac₂O) and DIEA inDMF; and (e1) washing the product formed in step (d1) with DMF.
 23. Themethod of claim 21 or claim 22 wherein performing an Fmoc deprotectionreaction followed by a coupling reaction comprises: (a2) treating theresin with a solution comprising piperidine in DMF; (b2) washing theresin with DMF; (c2) washing the resin with a solution comprising DMFand oxyma; (d2) contacting the resin with at least one Fmoc-protectedamino acid and a first amount of a solution comprisingdiisopropylcarbodiimide (DIC) and ethyl cyanohydroxyiminoacetate(oxyma); (e2) contacting the resin with a second amount of a solutioncomprising DIC and oxyma; and (f2) washing the product formed in step(e2) with DMF.
 24. The method of claim 23, wherein the resin iscontacted with the second amount of a solution comprising DIC and oxymaabout 30 minutes after contacting the resin with the first amount of asolution comprising DIC and oxyma.
 25. The method of claim 21 or claim22 wherein performing an Fmoc deprotection reaction followed by acoupling reaction comprises: (a2) treating the resin with a solutioncomprising piperidine and oxyma in DMF; (b2) washing the resin with DMF;(c2) washing the resin with a solution comprising DMF and oxyma; (d2)contacting the resin with at least one Fmoc-protected amino acid and afirst amount of a solution comprising diisopropylcarbodiimide (DIC) andethyl cyanohydroxyiminoacetate (oxyma); (e2) contacting the resin with asecond amount of a solution comprising DIC and oxyma; and (f2) washingthe product formed in step (e2) with DMF.
 26. The method of claim 25,wherein the resin is contacted with a first amount of a solutioncomprising piperidine and oxyma in DMF for 15 minutes followed bycontacting the resin with a second amount of a solution comprisingpiperidine and oxyma in DMF for 30 minutes.
 27. The method of any one ofclaims 23 to 26, wherein the at least one Fmoc-protected amino acid isFmoc-Gln(Trt)-Thr(ψ^(Me,Me)pro)-OH.
 28. The method of any one of claims23 to 26, wherein the at least one Fmoc-protected amino acid isFmoc-Gly(Tmb)-OH.
 29. The method of any one of claims 23 to 26, whereinthe at least one protected amino acid is Boc-His(Trt)-Gly-OH.
 30. Themethod of any one of claims 23-29, wherein the method further comprises,between steps (e2) and (f2), performing a coupling test, wherein thecoupling test is a Kaiser test.
 31. The methods of any one of claims16-30, wherein the GLP-2 analog peptide is apraglutide.
 32. Acomposition comprising the GLP-2 analog peptide produced using themethod of any of claims 16-31.
 33. A method of treating short bowelsyndrome associated intestinal failure (SBS-IF) or short bowel syndromeassociated intestinal insufficiency (SBS-II) in a subject comprisingadministering apraglutide, or pharmaceutically acceptable salt thereof,to the subject, wherein the apraglutide or pharmaceutically acceptablesalt thereof is administered at a dose of about 2.5 mg/week when thesubject has a body weight of less than 50 kg, or wherein the apraglutideor pharmaceutically acceptable salt thereof is administered at a dose ofabout 5 mg/week when the subject has a body weight greater than or equalto 50 kg.
 34. The method of claim 33, wherein the apraglutide, orpharmaceutically acceptable salt thereof, is administered bysubcutaneous injection.
 35. The method of claim 33 or claim 34, whereinthe subject has colon-in-continuity, and wherein the apraglutide, orpharmaceutically acceptable salt thereof is administered for about 48weeks.
 36. The method of claim 35, wherein the subject has greater than50% colon-in-continuity.
 37. The method of claim 33 or claim 34, whereinthe subject has at least one stoma, and wherein the apraglutide isadministered for about 24 weeks.
 38. The method of claim 33, wherein theadministration of apraglutide or a pharmaceutically acceptable saltthereof increases the intestinal absorption of dietary intake wet weightin a subject relative to an untreated or placebo treated subject. 39.The method of claim 33, wherein the administration of apraglutide or apharmaceutically acceptable salt thereof decreases fecal output in asubject relative to an untreated or placebo treated subject.
 40. Themethod of claim 33, wherein the administration of apraglutide or apharmaceutically acceptable salt thereof increases absolute urine volumeoutput in a subject relative to an untreated or placebo treated subject.41. The method of claim 33, wherein the administration of apraglutide ora pharmaceutically acceptable salt thereof increases intestinalabsorption of sodium and potassium in a subject relative to an untreatedor placebo treated subject.
 42. The method of claim 33, wherein theadministration of apraglutide or a pharmaceutically acceptable saltthereof increases sodium and potassium urine excretion in a subjectrelative to an untreated or placebo treated subject.
 43. The method ofclaim 33, wherein the administration of apraglutide or apharmaceutically acceptable salt thereof increases intestinal absorptionof energy in a subject relative to an untreated or placebo treatedsubject.
 44. The method of claim 33, wherein the administration ofapraglutide or a pharmaceutically acceptable salt thereof decreases theenergy content of fecal output in a subject relative to an untreated orplacebo treated subject.
 45. The method of claim 33, wherein theadministration of apraglutide or a pharmaceutically acceptable saltthereof increases intestinal absorption of carbohydrates, proteins, andlipids in a subject relative to an untreated or placebo treated subject.46. The method of claim 33, wherein the administration of apraglutide ora pharmaceutically acceptable salt thereof increases citrullineconcentration in a subject relative to an untreated or placebo treatedsubject.